Compounds and methods for modulating activation of NF-κB

ABSTRACT

Compositions and methods for modulating the activation of nuclear factor κB (NF-κB) are provided. The compositions comprise one or more agents that modulate ubiquitination of phosphorylated IκBα and/or IκBβ. Such compositions may be used for treating diseases associated with NF-κB activation. Modulating agents include human E3 ubiquitin ligases, antibodies thereto and variants thereof, as well as related proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Continuation of Ser. No. 09/210,060 filed Dec. 10, 1998 nowabandoned which is hereby incorporated in its entirety by reference.

TECHNICAL FIELD

The present invention relates generally to compositions and methods formodulating the activation of nuclear factor κB (NF-κB). The invention ismore particularly related to agents that modulate ubiquitination ofphosphorylated IκBα and/or IκBβ and to methods for treating diseasesassociated with NF-κB activation. Modulating agents encompassed by thepresent invention include E3 ubiquitin ligases, and portions andvariants thereof.

BACKGROUND OF THE INVENTION

NF-κB is a transcription factor that plays a pivotal role in the highlyspecific pattern of gene expression observed for immune, inflammatoryand acute phase response genes, including interleukin 1, interleukin 8,tumor necrosis factor and certain cell adhesion molecules. Like othermembers of the Rel family of transcriptional activators NF-κB issequestered in an inactive form in the cytoplasm of most cell types. Avariety of extracellular stimuli including mitogens, cytokines,antigens, stress inducing agents, UV light and viral proteins initiate asignal transduction pathway that ultimately leads to NF-κB release andactivation.

Important modulators of NF-κB activation are the inhibitor proteins IκBαand IκBβ (referred to herein as IκB), which associate with (and therebyinactivate) NF-κB in the cytoplasm of nonstimulated cells. Activationand nuclear translocation of NF-κB occurs following signal-inducedphosphorylation of IκB, which leads to proteolysis via the ubiquitinpathway. For IκBα, the stimulus-induced phosphorylation at serines 32and 36 renders the inhibitor a target for ubiquitination at lysines 21and 22, resulting in degradation. Similarly, phosphorylation of IκBβ atserines 19 and 23 renders the inhibitor a target for ubiquitination atlysin 9. However. the component(s) of the ubiquitin system mediating IκBrecognition have not been identified.

Degradation of a protein via the ubiquitin pathway proceeds by twodiscrete and successive steps: (a) covalent attachment of multipleubiquitin molecules to the protein substrate, and (b) degradation of thetargeted protein by the 26S proteasome complex. The ubiquitin pathwayconsists of several components that act in concert and in a hierarchicalmanner (for reviews, see Ciechanover, Cell 79:13, 1994; Hochstrasser,Curr. Op. Cell. Biol. 7:215, 1995; Jentsch and Schlenker, Cell 82:881,1995; Deshaies, Trends Cell Biol. 5:428, 1995). One such component, asingle E1 enzyme, carries out activation of ubiquitin. Several majorspecies of E2 enzymes have been characterized in mammalian cells,plants, and yeast. E2 enzymes probably bind to the ligase E3 (Reiss andHersko, J. Biol. Chem. 265:3685, 1990; Dohmen et al., Proc. Natl. Acad.Sci. USA 88:7351, 1991) and it appears that each E2 enzyme can act withone or more E3 proteins (Nuber et al., J. Biol. Chem. 271:2795, 1996;Orian et al., J. Biol. Chem. 270:21707, 1995; Stancovski et al., Mol.Cell. Biol. 15:7106, 1995; Gonen et al., J. Biol. Chem. 271:302, 1996).

Only few E3 enzymes (ubiquitin ligases) have been described. MammalianE3α (UBR1 in yeast) and E3β recognize protein substrates via their freeN-terminal amino acid residues (“N-end rule”; Varshavsky, Cell 69:725,1992; Hershko and Ciechanover, Ann. Rev. Biochem. 61:761, 1992). Cdc53is probably an E3 involved in targeting phosphorylated G1 cyclins(Willems et al., Cell 86:453, 1996). E6-AP is involved in recognition ofp53 (Scheffner et al., Cell 75:495, 1993), and a series of unique E6-APhomologous proteins have been identified (Huibregtse et al., Proc. Natl.Acad. Sci. USA 92:2563, 1995): Nedd4 is involved the degradation of theepithelial Na⁺ channel (Staub et al, Embo J. 15:2371, 1996) and RSP5(NIP1) is involved in tagging the permeases Gap1 and Fur1 (Hein et al.,Mol. Microbiol. 18:77, 1995), whereas Pub1 targets Cdc25 (Nefsky andBeach, EMBO J. 15:1301, 1996). Several other E3 enzymes that have beenrecently isolated appear to be involved in the degradation of c-Fos, asubset of muscle proteins, and in the processing of p105, the NF-κBprecursor (Orian et al., J. Biol. Chem. 270:21707, 1995; Stancovski etal., Mol. Cell. Biol. 15:7106, 1995; Gonen et al., J. Biol. Chem.271:302, 1996). Thus, it appears that the ligases represent a large,mostly unraveled family of enzymes and, except for the mode ofrecognition of the “N-end rule” ligases (E3α and E3β), the recognitionmotifs of all other known substrates of the ubiquitin system have notbeen identified.

Accordingly, there is a need in the art for an improved understanding ofIκB degradation via the ubiquitin pathway, and for the identification ofmodulators of this degradation process for use in treating diseasesassociated with activation of NF-κB. The present invention fulfillsthese needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor modulating the activation of nuclear factor κB (NF-κB) by modulatingubiquitination of phosphorylated IκBα and/or IκBβ. Within one aspect,the present invention provides isolated human E3 ubiquitin ligasepolypeptides. Such polypeptides may comprise a human E3 ubiquitin ligasesequence as recited in SEQ ID NO:16, or a portion or variant thereofthat differs in one or more amino acid substitutions, insertions,deletions and/or additions, such that the polypeptide (a) enhancesubiquitination of phosphorylated IκB or (b) binds to phosphorylated IκBand inhibits ubiquitination of phosphorylated IκB. Within certainembodiments, such a polypeptide may have the sequence recited in SEQ IDNO:16 or a variant thereof that differs in one or more amino aciddeletions, insertions or substitutions at no more than 20% of the aminoacid residues in SEQ ID NO:16, such that the polypeptide enhancesubiquitination of phosphorylated IκB. Within further embodiments, such apolypeptide may comprise a portion of a human E3 ubiquitin ligase, orvariant of such a portion, wherein the portion binds to phosphorylatedIκB and inhibits ubiquitination of phosphorylated IκB.

The present invention further provides, within other aspects, isolatedpolynucleotides that encode a polypeptide as described above. Withincertain embodiments, such polynucleotides may encode a portion of ahuman E3 ubiquitin ligase, or variant of such a portion, as describedabove. Antisense polynucleotides comprising at least 10 consecutivenucleotides complementary to such a polynucleotide are also provided.Expression vectors comprising such a polynucleotide, and host cellstransformed or transfected with such an expression vector, are furtherprovided.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove in combination with a physiologically acceptable carrier.

Within other aspects, the present invention provides isolatedantibodies, and antigen binding fragments thereof, that bind to a humanE3 ubiquitin ligase having a sequence recited in SEQ ID NO:16. Suchantibodies may be monoclonal.

Within further aspects, pharmaceutical compositions are provided,comprising an antibody or fragment thereof as described above incombination with a physiologically acceptable carrier.

The present invention further provides methods for modulating NF-κBactivity in a patient, comprising administering to a patient apharmaceutical composition as described above.

Within further aspects, the present invention provides methods fortreating a patient afflicted with a disorder associated with NF-κBactivation, comprising administering to a patient a therapeuticallyeffective amount of a pharmaceutical composition as described above, andthereby treating a disorder associated with NF-κB activation. Suchdisorders include inflammatory diseases, autoimmune diseases, cancer andviral infection.

Within further aspects, the present invention provides methods forscreening for an agent that modulates NF-κB activity, comprising thesteps of: (a) contacting a candidate agent with a human E3 ubiquitinligase polypeptide, wherein the polypeptide comprises a sequence recitedin SEQ ID NO:16 or a portion or variant thereof that differs in one ormore amino acid substitutions, insertions, deletions or additions, suchthat the polypeptide enhances ubiquitination of phosphorylated IκB,under conditions and for a time sufficient to permit interaction betweenthe polypeptide and candidate agent; and (b) subsequently evaluating theability of the polypeptide to enhance ubiquitination of phosphorylatedIκB, relative to a predetermined ability of the polypeptide to enhanceubiquitination of phosphorylated IκB in the absence of candidate agent;and therefrom identifying an agent that modulates NF-κB activity.Candidate agents for use within such screens include, but are notlimited to, small molecules present within a combinatorial library.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are autoradiograms depicting the results of SDS-PAGEanalysis of ubiquitination assays performed in the presence and absenceof various IκB E3 recognition motifs. Unless otherwise indicated, thesubstrate was an ³⁵S-labelled, HA-tagged IκB polypeptide that wasphosphorylated and NF-κB complex-associated.

In FIG. 1A, lane 1 shows the ubiquitination of an IκBα polypeptide thatcontains alanine residues at positions 32 and 36 (S32/36A; SEQ ID NO:13)and lane 2 shows the ubiquitination of a non-phosphorylated wild-typeIκBα polypeptide (SEQ ID NO:12). In lanes 3-14, the ubiquitinationsubstrate was wild-type IκBα (SEQ ID NO:12). In lane 3, ubiquitinationwas performed in the absence of ATP; and in lanes 4-14 the reaction wasperformed in the presence of ATPγS with (lanes 5-14) or without (lane 4)an IκB E3 recognition motif or other peptide. The peptides shown are:400 μM c-Fos phosphopeptide (ppFos (SEQ ID NO:10), lane 5); 400 μMserine 32, 36 to alanine substituted IκBα peptide (pp21S/A (SEQ IDNO:11), lane 6); 401 μM doubly phosphorylated IκBα peptide (p21 (SEQ IDNO:9), lane 7); 400 μM non-phosphorylated IκBα peptide (p21 (SEQ IDNO:9), lane 8); 100 μM singly phosphorylated IκBα peptides (ppS32 (SEQID NO:9), lane 9; ppS36 (SEQ ID NO:9), lane 10); and 40 μM shorter,doubly phosphorylated IκBα peptides (pp19 (SEQ ID NO:8), lane 11); pp15(SEQ ID NO:7), lane 12; pp11 (SEQ ID NO:6), lane 13; pp7 (SEQ ID NO:5),lane 14).

In FIG. 1B, the ubiquitination substrate was free wild type IκBα (SEQ IDNO:12, lanes 1-3) or free S32/36A substituted IκBα (SEQ ID NO:13, lanes4-6). The reaction was performed in the absence (lanes 1 and 4) orpresence (lanes 2, 3, 5 and 6) of ATPγS. 40 μM doubly phosphorylatedIκBα peptide (pp21 (SEQ ID NO:9) was added to the conjugation reactionmixture in the samples shown in lanes 3 and 6.

In FIG. 1C, the ubiquitination of bulk cellular proteins in HeLa extractis shown. Lane 1 shows the ubiquitination in the absence of ATP, andlane 5 shows the ubiquitination in the presence of ATP. In lanes 3-5, anIκB E3 recognition motif or other peptide was added: 40 μM doublyphosphorylated IκBα peptide (pp21 (SEQ ID NO:9), lane 2); 400 μM c-Fosphosphopeptide (ppFos (SEQ ID NO:10), lane 3); and 400 μMnon-phosphorylated IκBα peptide (p21 (SEQ ID NO:9), lane 4).

In FIG. 1D, the ubiquitination substrate was phosphorylated (lanes 2-7)or non-phosphorylated (lane 1) wild type IκBβ (SEQ ID NO:14). Reactionswere performed in the absence (lane 2) or presence (lanes 1, 3-7) ofATPγS, and with (lanes 4-7) or without (lanes 1-3) an IκB E3 recognitionmotif or other peptide. The peptides shown are: 40 μM doublyphosphorylated IκBβ peptide (pp21 (SEQ ID NO:9), lane 4); 400 μM c-Fosphosphopeptide (ppFos (SEQ ID NO:10), lane 5); 40 μM doublyphosphorylated IκBα peptide (pp19 (SEQ ID NO:8), lane 6); and 400 μMnon-phosphorylated IκBα peptide (p21 (SEQ ID NO:9), lane 7).

FIG. 2 is an autoradiogram depicting the results of an in vitroubiquitin-dependent degradation assay performed using extracts fromstimulated HeLa cells. In each lane of the SDS-PAGE, the level ofphosphorylated (upper band) and non-phosphorylated (lower band)HA-tagged IκBα polypeptide (SEQ ID NO:12) following the degradationassay is shown. Lane 1 shows the level of these polypeptides following adegradation assay performed without ATP. In lanes 2-6, ATP was includedin the reaction mixture. 40 μM candidate modulating agents were added tothe reactions shown in lanes 3-6: doubly phosphorylated IκBα peptide(pp21 (SEQ ID NO:9), lane 3); doubly phosphorylated IκBα peptide (pp19(SEQ ID NO:8), lane 4); c-Fos phosphopeptide (ppFos (SEQ ID NO:10), lane5); and non-phosphorylated IκBα peptide (p21 (SEQ ID NO:9), lane 6).

FIG. 3A is an autoradiogram depicting the results of SDS-PAGE analysisof ubiquitination assays performed using flow-through fractions of HeLacell lysate fractionated over modulating agent columns. In each case,the substrate was a ³⁵S-labelled, HA-tagged IκBα polypeptide (SEQ IDNO:12) that was phosphorylated and NF-κB complex-associated. Lane 1shows the level of ubiquitination using a non-fractionated extract. Inlanes 2-9, the extract was fractionated over a peptide-Sepharose®column. The peptides used were: c-Fos phosphopeptide (ppFos (SEQ IDNO:10), lane 2); serine 32, 36 to alanine substituted IκBα peptide(pp21S/A (SEQ ID NO:11), lane 3); doubly phosphorylated IκBα peptide(pp21 (SEQ ID NO:9), lanes 4-6); and doubly phosphorylated IκBα peptide(pp19 (SEQ ID NO:8), lanes 7-9). In addition, reticulocyte Fraction II(160 μg) was added to the ubiquitination reactions shown in lanes 5 and8, and Fraction I (160 μg) was added to the reactions in lanes 6 and 9.

FIG. 3B is an autoradiogram showing the ubiquitination of bulk cellularproteins in HeLa extract. Lane 1 shows the ubiquitination in the absenceof ATP, and lane 2 shows the ubiquitination in the presence of ATP, butwithout candidate modulating agent. In lanes 3-6, candidate modulatingagents were added: 40 μM doubly phosphorylated IκBα peptide (pp19 (SEQID NO:8), lane 3); 400 μM c-Fos phosphopeptide (ppFos (SEQ ID NO:10),lane 4); 400 μM serine 32, 36 to alanine substituted IκBα peptide(pp21S/A (SEQ ID NO:11), lane 5); and 40 μM doubly phosphorylated IκBαpeptide (pp21 (SEQ ID NO:9), lane 6).

FIGS. 4A-4F are micrographs showing the effect of candidate modulatingagents on nuclear NF-κB translocation. In FIGS. 4A-C, pp21 (FIGS. 4A and4B) or ppFos (FIG. 4C) was microinjected into the cytoplasm of HeLacells. Cells were then activated immediately with TNFα and immunostainedwith anti-p65 antibodies. In FIGS. 4D-F, pp21 (FIG. 4D) or ppFos (FIG.4F) was injected into the cytoplasm of human vascular endothelial cells(HUVEC). Cells were then activated immediately with TNFα andimmunostained with anti-E-selectin antibodies. FIG. 4E is a phasecontrast photograph of FIG. 4D. In each micrograph, the injected cellsare marked by large arrows. A non-injected, E-selectin negative cell ismarked by a small arrow in FIGS. 4D and 4E.

FIGS. 4G and 4H are graphs presenting a summary of the microinjectionexperiments shown in FIGS. 4A-4F. In FIG. 4G, the percent of HeLa cellsdisplaying nuclear p65 staining is shown. 90 and 42 cells weremicroinjected with pp21 and ppFos, respectively. FIG. 4H shows thepercent of HUVEC displaying E-selectin staining. 160 and 36 cells weremicroinjected with pp21 and ppFos, respectively. For each graph, column1 shows the level in the absence of an IκB E3 recognition motif or otherpeptide and TNFα activation. Columns 2-4 show the level following TNFαactivation in the absence of peptide (column 2) or in the presence ofpp21 (column 3) or ppFos (column 4).

FIG. 5 is an autoradiogram depicting the results of a Western blotanalysis showing the immunoprecipitation of pIκBα-associatedubiquitin-ligase activity from TNFα-activated cells. The pIκBα/NF-κBcomplex was immunoprecipitated from proteasome-inhibited,TNFα-stimulated or non-stimulated HeLa cells and subjected to in vitroubiquitination upon addition of ubiquitin, ATP-γS and the followingcomponents: lane 1, UBC5C; lane 2, UBC5C and E1, lane 3, none; lanes4-6, UBC5C and E1 as indicated; lane 7. UBC5C, E1 and pIκBα-peptide;lane 8, UBC5C, E1 and serine-substituted IκBα peptide; lane 9, a sampleof TNFα-stimulated HeLa lysate. Cell-stimulation is indicated in theTNFα row. Monomeric and ubiquitin-conjugated IκBα are marked at theleft, bottom and top of the figure.

FIG. 6 is an autoradiogram illustrating the association of theubiquitin-ligase with the IκBα NF-κB complex, followingIKK-phosphorylation of IκBα at the DSGLDS (SEQ ID NOs:8 and 19) site.³⁵S-labeled IκBα/NF-κB complex immunopurified from non-activated cellswas phosphorylated by IKK-2EE (where marked by + at the top), incubatedwith non-activated HeLa lysate as an E3 source, washed and subjected toin vitro ubiquitination in the presence of ATPγS, ubiquitin, E1, UBC5C(except where an excluded component is indicated by Abst Ub-Enz). Lanes2-7 show phosphorylation by IKK; lanes 1 and 3-7 show the effect ofincubation with HeLa lysate; in lane 4, a pIκBα peptide was added duringthe incubation with HeLa lysate: in lane 5, serine-substituted IκBαpeptide was added during HeLa incubation; in lane 6, E1 was omitted fromthe ubiquitination stage; and in lane 7, UBC5C was omitted duringubiquitination.

FIGS. 7A and 7B illustrate the identification of IκBα-binding proteinsassociated with ubiquitin-ligase activity. FIG. 7A is a photographshowing Colloidal Blue staining of SDS-polyacrylamide gel samples ofimmunopurified fractions containing IκBα/NF-κB and associated proteins.IκBα/NF-κB complex was phosphorylated by IKK-2EE (lanes 2, 3) ormock-phosphorylated and used to adsorb the ubiquitin-ligase from HeLalysate (lanes 1, 2). Molecular-size markers (κD) are indicated on theright. Proteins identified by mass-spectrometry analysis are indicatedon the left. Gel-sites corresponding to the bands associated with theubiquitin-ligase activity (p54 and p58) are marked on the left bybrackets. FIG. 7B is an autoradiogram of proteins adsorbed ontopIκBα/NF-κB from ³⁵S-labeled HeLa cells. Radiolabeled HeLa lysate wasincubated with IKK-phosphorylated antibody-immobilized IκBα/NF-κBcomplex. The immune-complexes were then washed, eluted and analyzed bySDS-PAGE and autoradiography. Lane 1 shows non-phosphorylated IκBα/NF-κBcomplex incubated with HeLa lysate; lanes 2-4 show phosphorylatedIκBα/NF-κB-complex incubated with HeLa lysate in the absence (lane 2) orpresence of pIκBα-peptide (lane 3) or serine-substituted IκBα-peptide(lane 4). Indicated on the left are molecular size markers (κD), Rel Aand IκBα bands; indicated in the right are the four pIκBα-associatedbands, three of which were displaced by the pIκBα peptide (arrows).

FIGS. 8A-8D show the results of a mass-spectrum analysis ofubiquitin-ligase associated p54. FIG. 8A shows a nanoelectrospray massspectrum of the unseparated tryptic peptide mixture from the 54 κD gelband excised from a ligase-positive lane (equivalent to lane 2 in FIG.7B). Peaks marked by arrows were fragmented and identified as peptidesderived from β-TrCP. The bar indicates the region enlarged in C. FIGS.8B and 8C present a comparison of the nanoelectrospray spectra of the 54κD band associated with (C) and without (B) ubiquitin-ligase activity.The peptide at m/z 714.38 was selected for sequencing. FIG. 8D is afragmentation spectrum of the peptide identified in FIG. 8C. A sequencetag was assembled from a series of doubly charged fragment ions andsearched in the nrdb data-base for a matching pattern. Fragment massescalculated for the retrieved β-TrCP sequence AAVNVVDFDDKYIVSASGDR (SEQID NO:20) were compared with the complete fragmentation spectrum toconfirm the match. Peaks matching expected fragment ions are marked bycircles.

FIGS. 9A and 9B present the sequence of a polynucleotide encoding ahuman E3 ubiquitin ligase (SEQ ID NO:15).

FIG. 10 presents a human E3 ubiquitin ligase protein sequence (SEQ IDNO:16).

FIGS. 11A-11C are Western blots illustrating binding and ubiquitinationspecificity of E3 ubiquitin ligase family members. Within these figures,mβ-TrCP indicates mouse β-TrCP, hβ-TrCP indicates human β-TrCP, Δβ-TrCPindicates human β-TrCP with a deletion of the F box region and Slimbindicates the Drosophila Slimb protein. FIG. 11A illustrates selectivebinding to pIκBα. Proteins were immunoprecipitated through a FLAGepitope from transfected 293T cells, incubated with immunopurifiedIκBα/NF-κB complex, which had been treated (−/+IKK) as indicated and thebound material was analyzed by Western blotting with the indicatedantibodies. The top panel shows specific pIκBα binding; the middle panelshows 10% of the substrate flow-through; the bottom panel is a blot ofthe immunoprecipitated proteins; and molecular size markers (KD) areindicated on the left. FIG. 11B shows that β-TrCP-pIκBα binding isabrogated by a phosphopeptide representing the pIκBα degradation motif(pp10), but not by a related non-phosphorylated peptide (pS/A). FIG. 11Cillustrates in vitro ubiquitination of pIκBα by the E3 family memberproteins. Immunopurified FLAG-tagged proteins were incubated with³⁵S-labeled IκBα/NF-κB complexes, treated (−/+IKK) as indicated andsubject to ubiquitination in the presence of ATPγS, ubiquitin, E1 andUBC5C. The IκBα substrate (composed of full-length and two degradationproducts), pIκBα-polyubiquitin conjugates and molecular size markers areindicated on the left.

FIGS. 12A and 12B illustrate inhibition of IκBα degradation and NF-κBactivation by overexpression of Δβ-TrCP, a dominant negative molecule.FIG. 12A is a graph depicting the results of a κB-dependent luciferaseassay in P/I-stimulated Jurkat cells transfected with κB-Luc reporterplasmid and the indicated expression vectors (i.e., from left to right,vector alone, vector encoding human β-TrCP, vector encoding human β-TrCPwith a deletion of the F box region and vector encoding Drosophila Slimbprotein). NF-κB activity is shown as relative (fold) luciferaseactivity, the non-stimulated empty FLAG vector being the reference(single-fold). FIG. 12B depicts the results of western blot analysis ofIκBα of phorbol-ester and Ca⁺⁺ ionophore [P/I]-stimulated andnon-stimulated Jurkat cells transfected with an empty FLAG vector orΔβ-TrCP. The post-stimulation interval (min) is indicated.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods useful for modulating the activation of nuclearfactor κB (NF-κB) and for treating, diseases associated withsuch-activation. In particular, the invention is directed to agents thatmodulate ubiquitination of phosphorylated IκB (i.e., IκBα and/or IκBβ).Such ubiquitination results in the release and activation of NF-κB.

The present invention is based, in part, on the identification andcharacterization of a human E3 ubiquitin ligase that recognizesphosphorylated and NF-κB-associated IκB. Polypeptides comprising this E3ubiquitin ligase, as well as portions and other variants thereof, may beused to modulate NF-κB activity in vitro or in a patient. Suchpolypeptides may also be used, for example, to identify agents (such assmall molecules) that may be used to modulate NF-κB activity, and totreat disorders associated with abnormal NF-κB activation.

HUMAN E3 UBIQUITIN LIGASE POLYPEPTIDES AND POLYNUCLEOTIDES

It has been found, within the context of the present invention, that ahuman E3 ubiquitin ligase that migrates as a 54 kD protein binds to, andenhances ubiquitination of, phosphorylated IκBα (phosphorylated IκBα isalso designated herein as pIκBα). The sequence of a polynucleotideencoding a human E3 ubiquitin ligase is provided in FIG. 9 and SEQ IDNO:15; and a full length human E3 ubiquitin ligase protein sequence isprovided in FIG. 10 and SEQ ID NO:16. Human E3 ubiquitin ligase has alsobeen found, within the context of the present invention, to be a memberof a family of F-box/WD proteins that includes β-TrCP (Margottin et al.,Mol. Cell 1:565-574, 1998) and the Drosophila Slimb protein (see Jiangand Struhl, Nature 391:493-496, 1998). As described in greater detailbelow, other members of this family share certain properties of E3, andsuch proteins and variants thereof may be used within certain methodsprovided herein for E3.

Human E3 ubiquitin ligase polypeptides encompassed by the presentinvention include native human E3 ubiquitin ligase (also referred toherein as “E3”), as well as portions and other variants thereof.Variants of E3 may differ in sequence from native E3 due to one or moreamino acid substitutions, deletions, additions and/or insertions, asdescribed herein, provided that the variant binds to and enhancesubiquitination of an IκB polypeptide as described herein. Preferably, avariant of E3 contains amino acid substitutions at no more than 20%,preferably no more than 15% and more preferably no more than 10%, of theresidues recited in SEQ ID NO:16. Variants further include truncatedpolypeptides and polypeptides containing additional amino acid sequencesthat have minimal influence on the activity of the polypeptide. A humanE3 ubiquitin ligase polypeptide may be of any length, provided that itretains the recited properties. In other words, such a polypeptide maybe an oligopeptide (i.e., consisting of a relatively small number ofamino acid residues, such as 8-10 residues, joined by peptide bonds), afull length protein (or variant thereof) or a polypeptide ofintermediate size (e.g., 20, 50, 200 or 400 amino acid residues).

Certain variants contain conservative substitutions. A “conservativesubstitution” is one in which an amino acid is substituted for anotheramino acid that has similar properties, such that one skilled in the artof peptide chemistry would expect the secondary structure andhydropathic nature of the polypeptide to be substantially unchanged.Amino acid substitutions may generally be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. Variants may also (oralternatively) be modified by, for example, the deletion or addition ofamino acids that have minimal influence on the immunogenicity, secondarystructure and hydropathic nature of the polypeptide.

As noted above, certain E3 polypeptides may contain additional aminoacid sequences at the amino and/or carboxy termini. For example, an E3sequence may be conjugated to a signal (or leader) sequence at theN-terminal end of the protein which co-translationally orpost-translationally directs transfer of the protein. A polypeptide mayalso, or alternatively, be conjugated to a linker or other sequence forease of synthesis, purification or identification of the polypeptide(e.g., poly-His), or to enhance binding of the polypeptide to a solidsupport. For example, a polypeptide may be conjugated to animmunoglobulin Fc region.

The ability of an E3 polypeptide to bind to phosphorylated IκB may bereadily determined using any binding assay known to those of ordinaryskill in the art. For example, pIκBα/NF-κB complexes may be incubatedwith immobilized E3 polypeptide, and the level of IκBα binding evaluatedusing anti-IκBα antibodies (in, for example, a Western blot). Withinsuch assays, an E3 polypeptide should bind detectably to the IκBα;preferably the E3 polypeptide binds at a level that is not substantiallydiminished relative to the native human E3. In other words, the abilityof a variant to bind detectably to phosphorylated and complexed IκBβ maybe enhanced or unchanged, relative to the native polypeptide, or may bediminished by less than 50%, and preferably less than 20%, relative tothe native polypeptide. It will be apparent that other suitablesubstrates may be substituted for pIκBα/NF-κB complexes within suchassays.

The ability of an E3 polypeptide to enhance ubiquitination ofphosphorylated IκB may be assessed by incubating the polypeptide withIκBα/NF-κB complex, along with ATPγS, ubiquitin E1 and ubiquitin E2, anddetecting the slow-moving IκBα-ubiquitin conjugates by Western blotusing IκBα-specific antibodies, as described herein. In general, an E3polypeptide should result in a detectable level of ubiquitination withinsuch an assay; preferably the level of ubiquitination is notsubstantially diminished relative to the level of ubiquitinationgenerated by a similar amount of native human E3.

Also encompassed by the present invention are polypeptides comprising aportion or other variant of E3 that retains the ability to bind tophosphorylated IκB, but does not retain the ability to enhanceubiquitination of IκB. Such polypeptides may be readily identified usingthe binding assays and ubiquitination assays provided herein, and maygenerally be used to inhibit ubiquitination of IκB. Such polypeptidesinclude those from which the F-box region (i.e., a region of the proteinthat interacts with one or more components of the ubiquitin cascade) hasbeen deleted. F box regions may generally be identified functionally(i.e., deletion of an F-box region results in a protein that fails torecruit appropriate components of the ubiquitin machinery) and based onthe present of an F-box region consensus sequence (see Patton et al.,Trends in Genet. 14:236-243, 1998). Certain such polypeptides contain adeletion of amino acids 122-168 of SEQ ID NO:16. Within certainembodiments, portions of E3 may comprise 10 to 374 consecutive aminoacid residues, preferably 50 to 250, consecutive amino acid residues ofthe sequence recited in SEQ ID NO:16.

The present invention further provides polynucleotides that encode an E3polypeptide as provided herein. Any polynucleotide that encodes such apolypeptide, or a portion or variant thereof as described herein, isencompassed by the present invention. Such polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Native DNA sequences encoding a human E3, or portion thereof, may beisolated using any of a variety of hybridization or amplificationtechniques, which are well known to those of ordinary skill in the art.Within such techniques, probes or primers may be designed based on theE3 sequence provided herein, and may be purchased or synthesized.Libraries from any suitable tissue may be screened. An amplified portionor partial cDNA molecule may then be used to isolate a full length genefrom a genomic DNA library or from a cDNA library, using well knowntechniques. Alternatively, a full length gene can be constructed frommultiple PCR fragments. Partial and full length polynucleotidescomprising such sequences, other portions of full lengthpolynucleotides, and sequences complementary to all or a portion of suchfull length molecules, are specifically encompassed by the presentinvention. In addition, homologues from other species are specificallycontemplated, and may generally be prepared as described herein.

Polynucleotide variants of the recited sequences may differ from anative E3 polynucleotide in one or more substitutions, deletions,insertions and/or additions. Preferred variants contain nucleotidesubstitutions, deletions, insertions and/or additions at no more than20%, preferably at no more than 10%, of the nucleotide positions.Certain variants are substantially homologous to a native gene, or aportion or complement thereof. Such polynucleotide variants are capableof hybridizing under moderately stringent conditions to a naturallyoccurring DNA sequence encoding an E3 protein (or a complementarysequence). Suitable moderately stringent conditions include prewashingin a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at50° C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS). Suchhybridizing DNA sequences are also within the scope of this invention.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.

As noted above, the present invention further provides antisensepolynucleotides and portions of any of the above sequences. Suchpolynucleotides may generally be prepared by any method known in the artincluding, for example, solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro or in vivotranscription of DNA sequences that are incorporated into a vectordownstream of a suitable RNA polymerase promoter (such as T3, T7 orSP6). Certain portions of a polynucleotide may be used to prepare anencoded polypeptide, as described herein. In addition, or alternatively,a portion may function as a probe (e.g., to detect E3 expression in asample), and may be labeled by a variety of reporter groups, such asradionuclides, fluorescent dyes and enzymes. Such portions arepreferably at least 10 nucleotides in length, and more preferably atleast 20 nucleotides in length. Within certain preferred embodiments, aportion for use as a probe comprises a sequence that is unique to an E3gene. A portion of a sequence complementary to a coding sequence (i.e.,an antisense polynucleotide) may also be used as a probe or to modulategene expression. DNA constructs that can be transcribed into antisenseRNA may also be introduced into cells or tissues to facilitate theproduction of antisense RNA.

Any polynucleotide may be further modified to increase stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkagesin the backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl- methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

Polynucleotides as described herein may be joined to a variety of othernucleotide sequences using established recombinant DNA techniques. Forexample, a polynucleotide may be cloned into any of a variety of cloningvectors, including plasmids, phagemids, lambda phage derivatives andcosmids. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors and sequencing vectors. Ingeneral, a vector will contain an origin of replication functional in atleast one organism, convenient restriction endonuclease sites and one ormore selectable markers. Additional initial, terminal and/or interveningDNA sequences that, for example, facilitate construction of readilyexpressed vectors may also be present. Suitable vectors may be obtainedcommercially or assembled from the sequences described by methodswell-known in the art. Other elements that may be present in a vectorwill depend upon the desired use, and will be apparent to those ofordinary skill in the art.

Vectors as described herein may generally be transfected into a suitablehost cell, such as a mammalian cell, by methods well-known in the art.Such methods include calcium phosphate precipitation, electroporationand microinjection.

E3 polypeptides may generally be prepared using standard automatedsynthesis techniques or by expression of recombinant DNA encoding thedesired polypeptide. In general, peptides may be prepared syntheticallyusing standard techniques, incorporating amino acids and/or amino acidanalogs. During synthesis, active groups of amino acids and/or aminoacid analogs may be protected as necessary using, for example, at-butyldicarbonate (t-BOC) group or a fluorenylmethoxy carbonyl (FMOC)group. Amino acids and amino acid analogs may be purchased commercially(e.g., Sigma Chemical Co.; Advanced Chemtec) or synthesized usingmethods known in the art. Peptides may be synthesized using a solidphase method, in which the peptides are attached to a resin such as4-methylbenzhydrylamine (MBHA), 4-(oxymethyl)-phenylacetamido methyl-and 4-(hydroxymethyl)phenoxy methyl-copoly(styrene-1% divinylbenzene)(Wang resin), all of which are commercially available, or top-nitrobenzophenone oxime polymer (oxime resin) which can be synthesizedas described by De Grado and Kaiser. J. Org. Chem. 47:3258, 1982. Thoseskilled in the art will realize that the choice of amino acids and/oramino acid analogs will depend, in part, on the specific physical,chemical or biological characteristics desired. Such characteristics aredetermined, in part, by the method of administration and the targetlocation within a patient.

Selective modification of the reactive groups in a peptide can alsoimpart desirable characteristics. Peptides can be manipulated whilestill attached to the resin to obtain N-terminal modified compounds suchas an acetylated peptide or can be removed from the resin using hydrogenfluoride or an equivalent cleaving agent and then modified. Compoundssynthesized containing the C-terminal carboxy group (Wang resin) can bemodified after cleavage from the resin or, in some cases, prior tosolution phase synthesis. Methods for modifying the N-terminus orC-terminus of a peptide are well known in the art and include, forexample, methods for acetylation of the N-terminus or amidation of theC-terminus. Similarly, methods for modifying side chains of the aminoacids or amino acid analogs are well known to those skilled in the artof peptide synthesis. The choice of modifications made to reactivegroups present on the peptide will be determined by the desiredcharacteristics.

An E3 polypeptide may also be a cyclic peptide. A cyclic peptide can beobtained by inducing the formation of a covalent bond between, forexample, the amino group at the N-terminus of the peptide and thecarboxyl group at the C-terminus. Alternatively, a cyclic peptide can beobtained by forming a covalent bond between a terminal reactive groupand a reactive amino acid side chain or between two reactive sidechains. It will be apparent to those of skill in the art that a cyclicpeptide is selected based on the desired properties. For example, acyclic peptide may provide increased stability, increased solubility,decreased immunogenicity or decreased clearance in vivo.

A newly synthesized peptide can be purified using a method such asreverse phase high performance liquid chromatography (RP-HPLC) or othermethods of separation based on size or charge. Furthermore, a purifiedpeptide can be characterized using these and other well known methodssuch as amino acid analysis and mass spectrometry.

Alternatively, polypeptides may generally be prepared from nucleic acidencoding the desired polypeptide using well known techniques. To preparean endogenous protein, an isolated cDNA may be used. To prepare avariant polypeptide, standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis may be used, andsections of the DNA sequence may be removed to permit preparation oftruncated polypeptides.

In general, any of a variety of expression vectors known to those ofordinary skill in the art may be employed to express recombinantpolypeptides of this invention. Expression may be achieved in anyappropriate host cell that has been transformed or transfected with anexpression vector containing a DNA sequence that encodes a recombinantpolypeptide. Suitable host cells include prokaryotes, yeast,baculovirus-infected insect cells and animal cells. Followingexpression, supernatants from host/vector systems which secreterecombinant protein or polypeptide into culture media may be firstconcentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. One or morereverse phase HPLC steps can be employed to further purify a recombinantpolypeptide.

In general, polypeptides and polynucleotides as described herein areisolated. An “isolated” polypeptide or polynucleotide is one that isremoved from its original environment. For example, anaturally-occurring protein is isolated if it is separated from some orall of the coexisting materials in the natural system. Preferably,polypeptides provided herein-are isolated to a purity of at least 80% byweight, more preferably to a purity of at least 95% by weight, and mostpreferably to a purity of at least 99% by weight. In general, suchpurification may be achieved using, for example, the standard techniquesof ammonium sulfate fractionation, SDS-PAGE electrophoresis, andaffinity chromatography. A polynucleotide is considered to be isolatedif, for example, it is cloned into a vector that is not a part of thenatural environment.

Antibodies

The present invention further provides antibodies, and antigen-bindingfragments thereof, that specifically bind to an E3 polypeptide. As usedherein, an antibody, or antigen-binding fragment, is said to“specifically bind” to a polypeptide if it reacts at a detectable level(within, for example, an ELISA) with the polypeptide, and does not reactdetectably with unrelated proteins. Antibodies may be polyclonal ormonoclonal. Preferred antibodies are those antibodies that inhibit orblock E3 activity and within a ubiquitination assay as described herein.Other preferred antibodies (which may be used, for example, inimmunokinase assays) are those that immunoprecipitate active E3, asdetermined using any standard assay, such as an assay provided herein.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In one such technique, an immunogen comprising the polypeptide isinitially injected into a suitable animal (e.g., mice, rats, rabbits,sheep and goats), preferably according to a predetermined scheduleincorporating one or more booster immunizations, and the animals arebled periodically. Polyclonal antibodies specific for the polypeptidemay then be purified from such antisera by, for example, affinitychromatography using the polypeptide coupled to a suitable solidsupport.

Monoclonal antibodies may be prepared, for example, using the techniqueof Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, andimprovements thereto. Briefly, these methods involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity (i.e., reactivity with the polypeptide of interest). Suchcell lines may be produced, for example, from spleen cells obtained froman animal immunized as described above. The spleen cells are thenimmortalized by, for example, fusion with a myeloma cell fusion partner,preferably one that is syngeneic with the immunized animal. For example,the spleen cells and myeloma cells may be combined with a nonionicdetergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) and digested by papain to yield Fab andFc fragments. The Fab and Fc fragments may be separated by, for example,affinity chromatography on protein A bead columns.

Ubiquitination Assays

As noted above, the ability of an E3 polypeptide to modulateubiquitination of phosphorylated IκB may be assessed by incubating thepolypeptide with IκBα/NF-κB complex (or any other suitable substrate),along with ATPγS, ubiquitin E1 and ubiquitin E2, and detectingIκBα-ubiquitin conjugates by, for example, Western blot usingIκBα-specific antibodies. IκB polypeptides for use in a ubiquitinationassay as described herein may be native human IκBα (SEQ ID NO:1) or IκBβ(SEQ ID NO:3), or may be a variant of a native protein. Polypeptidevariants of IκB are generally modified such that the ability of thevariant to be phosphorylated and ubiquitinated within a ubiquitinationassay as described herein is not substantially diminished. An IκBpolypeptide may be labeled. For example, 34S may be incorporated into aIκB polypeptide by in vitro translation of the polypeptide in thepresence of ³⁵S-methionine, using standard techniques.

An IκB polypeptide may generally be prepared from DNA encoding thepolypeptide by expression of the DNA in cultured host cells or bytranslation using an in vitro system such as wheat germ extract. If hostcells are employed, such cells are preferably are bacteria, yeast,baculovirus-infected insect cells or mammalian cells. The recombinantDNA may be cloned into any expression vector suitable for use within thehost cell, using techniques well known to those of ordinary skill in theart. In vitro translation of polypeptide may generally be performedaccording to the manufacturer's instructions.

Expressed IκB polypeptides may be used without purification following invitro translation. Alternatively, a polypeptide may be isolated insubstantially pure form. An IκB polypeptide may be isolated to a purityof at least 80% by weight, preferably to a purity of at least 95% byweight, and more preferably to a purity of at least 99% by weight. Ingeneral, such purification may be achieved using, for example, therepresentative purification method described herein or the standardtechniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis,and affinity chromatography.

Certain ubiquitination assays may employ a cellular E3 to characterizemodulators of E3 activity. Within such assays, cellular extracts fromstimulated or non-stimulated Jurkat, HeLa, THP-1 or endothelial cellsmay be incubated in vitro with an IκB polypeptide in the presence of ATPand the phosphatase inhibitor okadaic acid. Cellular extracts maygenerally be prepared according to the method of Alkalay et al., Proc.Natl. Acad. Sci. USA 92:10599, 1995. The incubation is performed underconditions sufficient to result in phosphorylation of the IκBpolypeptide (at serines 32 and 36 for IκBα and variants thereof) andassociation of the phosphorylated polypeptide (pIκB) with thecellular-derived NF-κB complex. For example, IκB polypeptide may beincubated with HeLa or Jurkat cell extract, ATP and okadaic acid.Incubation for 90 minutes at 30° C. is generally sufficient to allowphosphorylation of the IκB polypeptide. Following this incubation, thepIκB/NF-κB complex may be immunopurified with, for example, anti-p65antibodies and subjected to in vitro ubiquitination in a cell freesystem, as described by Alkalay et al., Proc. Natl. Acad. Sci. USA92:10599, 1995. The level of ubiquitination may then be evaluated usingthe well known techniques of SDS-PAGE, followed by autoradiography.

Under these conditions, a wild type ³⁵S-pIκBα polypeptide generatesmultiply ubiquitinated species in the presence of ATPγS (see FIG. 1A,lane 4). Neither ³⁵S-labeled S32/36A mutant of IκBα (lane 1), nor thenon-phosphorylated wild type ³⁵S-IκBα (lane 2) are ubiquitinated.However, free forms of either mutant or wild type IκBα are readilyconjugated (FIG. 1B). Similarly, a free (but not a complex-associatedlysine 21, 22 mutant of IκBα can be ubiquitinated in vitro. Thus, unlikeubiquitination assays performed using free IκB polypeptides, theubiquitination assay provided herein targets only IκB polypeptides thatare complex-associated and appropriately phosphorylated.

A ubiquitination assay as described above may be used to identify agentsthat modulate ubiquitination of IκB. Modulating agents may includeantibodies (e.g., monoclonal), peptides, small molecules (e.g., from acombinatorial library) and other drugs that stimulate or, preferably,inhibit ubiquitination of an IκBα and/or IκBβ polypeptide. In general,such agents may be identified by including a candidate modulating agentin the ubiquitination reaction, which may otherwise be performed asdescribed above, and evaluating the effect of the agent on the level ofubiquitination. A suitable concentration of candidate agent for use insuch an assay generally ranges from about 0.1 μM to about 1 mM. Forpeptide candidate agents, a peptidase inhibitor such as Bestatin (40μg/mL) may also be added, and the amount of peptide preferably rangesfrom about 10 μM to about 1 mM. A candidate agent that results in astatistically significant effect on the level of ubiquitination is amodulating agent encompassed by the present invention.

Agents may be further evaluated by microinjection of the agent (e.g.,about 5 mg/mL of a peptide agent) into a suitable cell (e.g., HeLa cellor primary human vascular endothelial cell). Following microinjection,cells may be stimulated (e.g., with TNFα) and incubated to allow NF-κBactivation. In HeLa cells, TNFα induces rapid nuclear translocation ofNF-κB into the nucleus, which may be detected by staining withp65-specific antibodies. Modulating agents induce a statisticallysignificant decrease in NF-κB translocation, and may reduce suchtranslocation to undetectable levels.

Primary human vascular endothelial cells (HUVEC) respond to TNFαstimulation by surface expression of NF-κB regulated adhesion proteinssuch as ICAM-1, V-CAM-1 and E-selectin (Read et al., Immunity2:493,1995; Chen et al., J. Immunol 155:3538, 1995). E-selectinexpression is particularly NF-κB dependent and is the major inducibleendothelial adhesion molecule for initial neutrophil attachment androlling on activated endothelium. Stimulated cells may be fixed andstained to detect expression of one or more NF-κB regulated adhesionproteins. Microinjection of a polypeptide or other modulating agentresults in a statistically significant inhibition of such expression,but does not affect the expression of NF-κB independent adhesionproteins, such as ICAM2.

Therapeutic Applications

As noted above, certain E3 polypeptides, polynucleotides, antibodies andother agents as described herein may generally be used as modulatingagents to specifically inhibit or enhance cellular NF-κB functions.Modulating agents may also be used to modulate ubiquitination of IκBαand/or IκBβ in a patient, thereby modulating NF-κB cellular function invivo. As used herein, a “patient” may be any mammal, including a human,and may be afflicted with a disease associated with NF-κB activation, ormay be free of detectable disease. Accordingly, the treatment may be ofan existing disease or may be prophylactic. Diseases associated withNF-κB activation include, but are not limited to, inflammatory diseases,autoimnune diseases, cancer and viral infection.

Treatment refers to administration of a modulating agent as describedherein. For administration to a patient, one or more such compounds aregenerally formulated as a pharmaceutical composition. A pharmaceuticalcomposition may be a sterile aqueous or non-aqueous solution, suspensionor emulsion, which additionally comprises a physiologically acceptablecarrier (i.e., a non-toxic material that does not interfere with theactivity of the active ingredient). Any suitable carrier known to thoseof ordinary skill in the art may be employed in the pharmaceuticalcompositions of the present invention. Representative carriers includephysiological saline solutions, gelatin, water, alcohols, natural orsynthetic oils, saccharide solutions, glycols, injectable organic esterssuch as ethyl oleate or a combination of such materials. Optionally, apharmaceutical composition may additionally contain preservatives and/orother additives such as, for example, antimicrobial agents,anti-oxidants, chelating agents and/or inert gases, and/or other activeingredients.

Alternatively, a pharmaceutical composition may comprise apolynucleotide encoding a modulating agent (such that the modulatingagent is generated in situ) in combination with a physiologicallyacceptable carrier. In such pharmaceutical compositions, thepolynucleotide may be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid, bacterial and viral expression systems, as well as colloidaldispersion systems, including liposomes. Appropriate nucleic acidexpression systems contain the necessary polynucleotide sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). DNA may also be “naked,” as described, for example, in Ulmer etal., Science 259:1745-1749, 1993.

Various viral vectors that can be used to introduce a nucleic acidsequence into the targeted patient's cells include, but are not limitedto, vaccinia or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors are wellknown to those of ordinary skill in the art. Preferably, the retroviralvector is a derivative of a murine or avianretrovirus including, but notlimited to, Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A retroviral vector may additionally transfer orincorporate a gene for a selectable marker (to aid in the identificationor selection of transduced cells) and/or a gene that encodes the ligandfor a receptor on a specific target cell (to render the vector targetspecific). For example, retroviral vectors can be made target specificby inserting a nucleotide sequence encoding a sugar, a glycolipid, or aprotein. Targeting may also be accomplished using an antibody, bymethods known to those of ordinary skill in the art.

Viral vectors are typically non-pathogenic (defective), replicationcompetent viruses, which require assistance in order to produceinfectious vector particles. This assistance can be provided, forexample, by using helper cell lines that contain plasmids that encodeall of the structural genes of the retrovirus under the control ofregulatory sequences within the LTR, but that are missing a nucleotidesequence which enables the packaging mechanism to recognize an RNAtranscript for encapsulation. Such helper cell lines include (but arenot limited to) Ψ2, PA317 and PA12. A retroviral vector introduced intosuch cells can be packaged and vector virion produced. The vectorvirions produced by this method can then be used to infect a tissue cellline, such as NIH 3T3 cells, to produce large quantities of chimericretroviral virions.

Another targeted delivery system for polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system for use as a delivery vehicle invitro and in vivo is a liposome (i.e., an artificial membrane vesicle).It has been shown that large unilamellar vesicles (LUV), which range insize from 0.2-4.0 μm can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci. 6:77, 1981). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques 6:882, 1988).

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity and may be, for example, organ-specific, cell-specific,and/or organelle-specific. Mechanistic targeting can be distinguishedbased upon whether it is passive or active. Passive targeting utilizesthe natural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

Routes and frequency of administration, as well doses, will vary frompatient to patient. In general, the pharmaceutical compositions may beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity or transdermally. Between 1 and 6 doses maybe administered daily. A suitable dose is an amount that is sufficientto show improvement in the symptoms of a patient afflicted with adisease associated with NF-κB activation. Such improvement may bedetected by monitoring inflammatory responses (e.g., edema, transplantrejection hypersensitivity) or through an improvement in clinicalsymptoms associated with the disease. In general, the amount ofmodulating agent present in a dose, or produced in situ by DNA presentin a dose, ranges from about 1 μg to about 100 mg per kg of host.Suitable dose sizes will vary with the size of the patient, but willtypically range from about 10 mL to about 500 mL for 10-60 kg animal.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Identification of an IκB E3 Recognition Motif UsingUbiquitination Assay

This Example illustrates a representative ubiquitination assay, and theuse of such an assay to evaluate peptides for the ability to inhibit IκBubiquitination.

A. In vitro Ubiquitination Assay

HA-tagged IκBα or HA-tagged IκBβ cDNAs (Haskill et al., Cell65:1281-1289, 1991) were translated in vitro in wheat germ extract inthe presence of ³⁵S-methionine according to the manufacturer'sinstructions (Promega, Madison, Wis.). To phosphorylate IκBα or IκBβ, 1μl of the extract containing the labeled protein was incubated for 90minutes at 30° C. in a reaction mixture having a final volume of 30 μl:100 μg HeLa or Jurkat cell extract (prepared as described by Alkalay etal., Proc. Natl. Acad. Sci. USA 92:10599, 1995), 2 mM ATP and 1 μMokadaic acid. During this incubation, the labeled IκB polypeptide wasphosphorylated at serines 32 and 36, and associated with the endogenousNF-κB complex (data not shown).

Following incubation, 1 μl of anti-p65 serum was added, and the NF-κBimmune complex was immobilized to Protein A-Sepharose® and subjected toin vitro ubiquitination in HeLa cell extract as described by Alkalay etal. Ubiquitinated proteins were separated by SDS-PAGE and visualized byautoradiography.

As shown in FIG. 1A, only wild type ³⁵S-pIκBα generated multiplyubiquitinated species (lane 4). Neither ³⁵S-labeled S32/36A mutant ofIκBα (lane 1) nor the non-phosphorylated wild type ³⁵S-IκBα (lane 2)were ubiquitinated, and no ubiquitination of pIκBα was seen in theabsence of ATP (lane 3).

The physiological relevance of this assay was further documented bycomparison of in vitro ubiquitination of free ³⁵S-IκB to that of acomplex-associated, phosphorylated substrate. Whereas acomplex-associated S32/36A mutant was not subject to ubiquitinconjugation in accordance with its in vivo fate, free forms of eithermutant or wild type IκBα were readily conjugated (FIG. 1B). Similarly,only free, but not a complex-associated lysine 21, 22 mutant of IκBαcould be ubiquitinated in vitro (data not shown). Thus, while the freeIκBα is recognized by the ubiquitin system in a non-discriminatorymanner, the complex-associated inhibitor is masked unless it isappropriately phosphorylated.

B. Identification of the IκBα-Ubiquitin Ligase Recognition Motif

To identify the IκBα-ubiquitin ligase recognition motif, variouspeptides were added at varying concentrations to the reaction mixturesin the presence of the peptidase inhibitor Bestatin (40 μg/ml). Thepeptides spanned the N-terminal signaling domain of the protein, andwere phosphorylated at one or both serine residues (32 and 36), or wereunmodified or serine-substituted. These peptides were included in theubiquitination reaction at different concentrations and tested forinhibition of pIκBα specific ubiquitination. When conjugation of freeIκBα was monitored, the translated protein was added directly to theconjugation reaction mixture.

Only peptides that were phosphorylated at both serine 32 and 36 (pIκBαpeptides) effectively inhibited pIκBα ubiquitination (FIG. 1A, lanes 7,11-14). A c-Fos phosphopeptide (ppFos, lane 5), a serine 32, 36 toalanine substituted IκBα peptide (p21 S/A, lane 6) and anon-phosphorylated peptide (p21, lane 8) had no detectable effect on theubiquitination of pIκB at a concentration of 400 μM. The IC₅₀ of thephosphorylated IκBα peptides were calculated and representativeinhibitory concentrations are shown in FIG. 1A. Doubly phosphorylatedIκBα peptides inhibited the pIκB conjugation reaction (lanes 7, 11-14)at an IC₅₀ of 5 μM. The sequences of these peptides are provided inTable I, above, and in SEQ ID NOs:5-9. In contrast, singlyphosphorylated peptides (lanes 9, 10) inhibited the pIκBα conjugation atan IC₅₀ of 400 μM. The minimal size peptide tested (pp7, lane 14),merely spanning the signaling phosphorylation site, was sufficient toeffectively inhibit the ubiquitination, although at somewhat higher IC₅₀(10 μM). Thus, a peptide comprising residues 21 to 41 of SEQ ID NO:1comprises a recognition domain for E3 ubiquitin ligase. Interestingly,lysine residues 21 and 22 are not essential for inhibition, implyingthat the ubiquitin-system recognition site is distinct from the actualconjugation site.

The specificity of the peptides was tested in two otherubiquitin-conjugation reactions: the conjugation of free wild type (FIG.1B lanes 1-3) or S32/36A mutant IκBα (FIG. 1B, lanes 4-6) and theubiquitin conjugation to the bulk of cellular proteins in HeLa extract(detected by ¹²⁵I-labeled ubiquitin according to Alkalay et al., FIG.1C). Neither reaction was affected by the addition of an IκBα-ubiquitinligase recognition motif or a control peptide.

Peptides comprising an IκBα-ubiquitin ligase recognition motif werefound to abolish the ubiquitination of the pIκBα related substrate pIκBβ(FIG. 1D). Similar to the conjugation of pIκBα, the specific conjugationof the IκBβ also required an associated NF-κB complex (not shown) andprior phosphorylation at the IκBα-homologous residues Ser 19 and 23. AnIκBβ substrate prepared in the absence of phosphatase inhibitors was notsubject to ubiquitination (FIG. 1D, lane 1). Peptides affected pIκBβubiquitination at an IC₅₀ that was similar to that observed for pIκBα(FIG. 1D, lanes 4-7). Hence, it appears that the same enzyme(s) targetboth IκBs for ubiquitin-dependent degradation.

The inhibitory pIκBα peptides were tested in a complementaryubiquitin-dependent in vitro degradation assay (Orian et al., J. Biol.Chem. 270:21707, 1995; Stancovski et al., Mol. Cell. Biol. 15:7106,1995). Using this assay, only pIκBα derived from stimulated cells isdegraded in vitro in a ubiquitin-dependent manner, whereas thenon-phosphorylated IκBα from the same cell extract is not subject todegradation. Incorporation of the conjugation-inhibitory phosphopeptidesinto the degradation assay resulted in stabilization of the pκBαsubstrate (FIG. 2, lanes 3, 4) whereas the non-phosphorylated peptideagent or a control phospho-Fos peptide had no effect on the specificpIκBα degradation (lanes 5, 6). Trimming the peptides at Lys 21/22 didnot diminish the degradation inhibitory effect (lane 4), indicating thatthe peptides do not abolish pIκBα degradation by exhausting theubiquitin-proteasome system as conjugatable substrates.

Example 2 Identification of Ubiquitin System Component Involved inSubstrate Recognition

This Example illustrates the identification of a specific E3 that isresponsible for recognition of pIκB polypeptides.

pIκBα-ubiquitin conjugation and degradation requires a full complementof the ubiquitin system enzymes: E1, a specific E2 derived from theubiquitin system fraction I, E2F1 (Alkalay et al., Proc. Natl. Acad.Sci. USA 92:10599, 1995; Chen et al., Cell 84:853, 1996) and a FractionII-component E3. To identify the ubiquitin system component involved inthe substrate recognition, HeLa lysate was fractionated over IκBβphosphopeptide columns, and the flow-through fractions were assayed forpIκBα conjugation. Peptides were coupled to NHS-Sepharose® (Pharmacia)according to the manufacturer's instructions at a concentration of 2mg/ml. 100 μg of HeLa extract were incubated with 2.5 μl coupled resinin the presence of 0.1% NP40 and 3% ovalbumin for 1 hour at 4° C. Theresin was discarded and the unbound material tested in theubiquitination assay described above.

Whereas a flow-through fraction from a control phosphopeptide column andan S32/36A peptide column retained full IκBα conjugation capacity (FIG.3A, lanes 2, 3) flow-through fractions from two different pIκBα peptideslost their IκBα specific conjugation capacity (lanes 4, 7). The depletedconjugating activity could be complemented by reticulocyte Fraction II(lanes 5, 8) that contains all the known species of E3 enzymes(Ciechanover, Cell 79:13, 1994). Complementation could not be obtainedby the addition of Fraction I or Fraction I and E1 (lanes 6 and 9,respectively), indicating that the peptide columns depleted an E3 ratherthan E2 or E1. Again, IκBα lysine residues 21 and 22 were dispensablefor retaining the E3 (compare FIG. 3A, lane 7 to lane 4), emphasizingthe distinction between the substrate recognition and conjugation site.The peptide column depletion was found to be specific for the IκB E3, asall flow-through fractions maintained full activity in random HeLaprotein conjugation (as detected by measuring the conjugation of ¹²⁵Iubiquitin, FIG. 3B). This indicates that a specific E3 is responsiblefor recognition of the pIκBs at the identified motif.

Example 3 Effect of Representative Peptides on Cellular NF-κB Activation

This Example illustrates the inhibition of cellular NF-κB activation bymicroinjection of peptides comprising an IκBα-ubiquitin ligaserecognition motif.

HeLa cells were plated on a grid coverslips (Cellocate, Eppendorf) 18hours before microinjection. Microinjection was performed with a 22amino acid pIκBα peptide (pp21; Table I and SEQ ID NO:9) or a controlphospho-Fos peptide (SEQ ID NO:10) using a semi-automated apparatus(Eppendorf). Peptides were injected into the cell cytoplasm at aconcentration of 5 mg/ml in 100 mM KCl, 5 mM Na₂HPO₄ (pH 7.2), andimmediately activated with TNFα (200 units/mL) for either 20 minutes(for NF-κB translocation) or 3 hours (for E-selectin expression).Following activation, the cells were fixed and stained with p65 specificantibodies (Mercurio et al., Genes & Dev. 7:705, 1993; Santa Cruz) ormonoclonal anti-E-selectin antibodies (R&D Systems).

In the absence of peptide, TNFα induces rapid nuclear translocation ofNF-κB into the nucleus, as shown by the p65 nuclear staining of 90% ofthe cells (see FIG. 4G, column 2). The pp21 peptide abolishedTNFα-stimulated NF-κB activation in 50%-70% of the microinjected cellsin several experiments (see representative fields in FIGS. 4A and 4B;and FIG. 4G, column 3). In contrast, the control pp-Fos peptide had noeffect on the rate of NF-κB induced nuclear translocation, as comparedto non-microinjected cells (FIGS. 4C and 4G, column 4).

To further assess the functional consequences of NF-κB inhibition, theIκB-E3 inhibitory peptide was microinjected into primary human vascularendothelial cells (HUVEC; Chen et al, J. Immunol 155:3538 1995). Thesecells respond to TNFα stimulation by surface expression of NF-κBregulated adhesion proteins, such as E-selectin. HUVEC cells wereplated, microinjected and stimulated as described above. Three hourspost stimulation the cells were fixed and stained for expression of theNF-κB dependent E-selectin. 75%-85% of the HUVEC cells were intenselystained for E-selectin following TNFα stimulation in severalexperiments. Microinjection of the pp21 peptide resulted in theinhibition of E-selectin expression in 70%-80% of the microinjectedcells (FIG. 4D; and FIG. 4H, column 3). In contrast, the control pp-Fospeptide had no effect on E-selectin expression, as compared tonon-microinjected cells (FIGS. 4F and 4H, column 4). Microinjection of acontrol, S32/36A substituted IκBα peptide had no effect on the rate ofE-selectin expression.

These results demonstrate that the subunit-specific degradation of thesignal-induced phosphorylated IκBα and IκBβ is mediated by a specificE3. The recognition domain for E3 ubiquitin ligase is a short sequence,centered around the two signal-acquired phosphoserines conserved in bothIκBs, representing the first biologically relevant E3 recognition motif.The specificity in IκB recognition is supported by the context of thephosphorylated substrate: an associated cellular complex masks thesubstrate from non-specific E3s. This feature restricts the NF-κBinhibitor degradation to the post-stimulation phase, at which it isexposed through site-specific phosphorylation event(s) to the specificligase. NF-κB activation and its resultant function can be specificallyabolished by in vivo inhibition of the IκB ligase, using a modulatingagent as provided herein.

Example 4 Further Characterization of IκBα Ubiquitination

This Example further illustrates the characterization of the ubiquitinligase associated with IκBα ubiquitination.

A. Cytokine Stimulation Promotes the Association Between pIκBα and aSpecific Ubiquitin-Ligase

To further study the recruitment of components of the ubiquitinmachinery by phosphorylated IκBα-complexes, pIκBα/NF-κB complexes werepurified from proteasome inhibited, TNF-α stimulated HeLa cells, andtheir ubiquitination potential was evaluated. HeLa cells werepre-incubated with the proteasome inhibitor ALLN (150 μM) for 1 hour andstimulated for 10 minutes with TNFα. IκBα/NF-κB complexes wereimmunoaffinity-purified with goat anti-Rel A (p65) antibodies (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.) and the cognate p65peptide (ELFPLIFPAEPAQASGP (SEQ ID NO:21), which was synthetic andpurchased from Alfa-Diagnostic, Inc., and then HPLC-purified, analyzedby mass spectrometry, verified for the predicted structure and proven tobe over 85% pure).

The immunopurified fraction was supplemented with various components ofthe ubiquitin system and subjected to in vitro ubiquitination. Inparticular the fraction was supplemented with 0.2 μg purified E1 and 1μg purified recombinant UBC5C (Jensen et al., J. Biol. Chem.270:30408-30414, 1995) and incubated for 90 minutes at 37° C. inreaction buffer containing: 50 mM Tris (pH 7.6), 2 mM MgCl₂, 1 mM DTT,20 nM okadaic acid, 1 mg/ml bovine ubiquitin (Sigma) and 5 mM ATPγS(Sigma). The reaction mix was then boiled in SDS-buffer and the sampleanalyzed by SDS-PAGE (8.5%) and phospho-imaging.

The addition of ubiquitin, purified E1 and a specific E2, UBC5C, wasfound to be sufficient to generate the full capacity IκBα-ubiquitinconjugating activity (FIG. 5, lane 2), evident in the accumulation ofhigh-molecular mass species that reacted with IκBα specific antisera.This activity was E1-dependent (compare lanes 1 and 2), and was notprovided by the corresponding immunopurified fraction fromnon-stimulated HeLa cells (compare lanes 4, 5, 6). As the stimulatedHeLa fraction contained both phosphorylated and non-phosphorylated IκBα,the observed conjugates could be derived from either IκB species.

To determine the source of the IκBα-conjugates, the ubiquitinationreactions were performed in the presence of a pIκBα peptide (pp12;CDRHDS[PO3]GLDS[PO3]; SEQ ID NO:22) (lane 7) or a serine/glutamic-acidsubstituted IκBα peptide (p12S/E) (lane 8). Both peptides weresynthetic, purchased from Alfa-Diagnostic, Inc., and then HPLC-purified,analyzed by mass spectrometry, verified for the predicted structure andproven to be over 85% pure. IκBα peptides were added at the indicatedconcentrations to the reaction mixtures in the presence of the peptidaseinhibitor Bestatin (40 μg/ml). Only pp12 abolished the formation ofpolyubiquitin-IκBα conjugates, indicating that ubiquitination wasspecific for pIκBα (Yaron et al., EMBO J. 16:6486-6494, 1997).

B. Phosphorylation is Necessary and Sufficient to Recruit SpecificUbiquitin-Ligase Activity

The finding that E1 and E2 specifically complemented pIκBα-conjugationof the stimulated HeLa fraction, but failed to complement anon-stimulated fraction, could be explained in several ways: a) HeLastimulation activates a specific pIκB-ubiquitin ligase, b) HeLastimulation modifies the substrate, thus rendering it liable toubiquitination, or c) HeLa stimulation is necessary for modifying boththe substrate and the ligase. To distinguish among these possibilities,a recombinant, constitutively active IKK2 protein (IKK2-EE) was used(Mercurio et al., Science 278:860-66, 1997). This protein phosphorylatesIκBα at serine 32/36 similarly to a TNFα activated IKK-complex.

Following immunoprecipitation of ³⁵S-labeled IκBα/NF-κB complexes from anon-stimulated HeLa lysate previously incubated with recombinant³⁵S-labeled IκBα, the complexes were phosphorylated by the recombinantIKK2-EE, eluted with the p65 cognate peptide and subjected to in vitroubiquitination. After incubation with 1KK2-EE, nearly all of the ³⁵S-IκBwas phosphorylated. Yet, the addition of ubiquitin, E1 and UBC5C did notresult in pIκBα phosphorylation (FIG. 6, lane 2). Therefore, IκBphosphorylation by IKK was not sufficient to promote its ubiquitinationin the presence of E1 and E1. Conceivably, pIκBα ubiquitination requiresan additional component of the HeLa lysate that was notco-immunopurified from non-stimulated cells.

To confirm this hypothesis, immuno-bound IκBα/NF-κB complexes wereincubated with a non-stimulated HeLa lysate, either directly orfollowing IKK2-EE phosphorylation, washed extensively with high-saltbuffer and eluted with the p65 peptide. Indeed, incubation of thephosphorylated IκB complexes (FIG. 6 lane 3), but not of thenon-phosphorylated ones (lane 1), with the HeLa lysate, provided thepIκB-ligase component(s) necessary for pIκBα conjugation. No signal wasobtained when E1 or E2 were omitted from the reaction, confirming thatthe signal at the top of the gel represents poly-ubiquitinIκBα-conjugates (lanes 5, 6). TNFα stimulated HeLa-lysate was notsuperior over a non-stimulated lysate in providing the necessary ligasecomponent.

The inhibitory effect of pp12 on pIκBα-ubiquitination (FIG. 5) suggestedthat the essential HeLa component associates specifically and stablywith the pIκBα recognition motif during the incubation period and laterfunctions in pIκB-ubiquitin conjugation. To test this assumption, weincluded in the incubation step pp12 or the control peptide p12S/E,which was removed together with the HeLa lysate, before eluting thefractions. The addition of pp12 (FIG. 6, lane 4), but not of p12S/E(lane 5), abrogated the ubiquitin-ligase activity associated with thepIκB-complex, while preserving the integrity of the substrate. This wasevident in the ability of the peptide-treated fractions to undergdoubiquitination in the presence of Reticulocyte Fraction II as an E3source (Alkalay et al., Mol. Cell Biol. 15:1294-301, 1995). Severalconclusions may be drawn from this experiment:

1) A ubiquitin-ligase component essential to pIκBα ubiquitination isrecruited by the IκBα/NF-κB complex from the HeLa lysate following IKKphosphorylation.

2) This conjugation-promoting component is contained in a non-stimulatedHeLa lysate, indicating that there is no need to activate theubiquitin-ligase by TNF-stimulation.

3) The essential ligase component is apparently specific and associateswith IκB through a direct interaction with the pIκB recognition motif(proved by pp12 inhibition of pIκBα-conjugation).

C. Isolation of the Specific Ubiquitin-Ligase Component that RecognizespIκBα

HeLa extract (250 mg) was incubated with 250 μl anti-p65 immunobeads.Following four washes in buffer A (1M KCl, 0.5% NP40, 50 mM Tris bufferpH 7.6, 1 mM DTT) and one wash in buffer B (50 mM Tris buffer, pH 7.6, 1mM DTT), half the beads were subject to in vitro phosphorylation withIKK and half underwent mock-phosphorylation. The beads were washed twicein buffer A and once in buffer B, agitated with 100 mg HeLa extract inthe presence of 1 μm okadaic acid for 30 min at 25° C., washed fourtimes with buffer A, once in buffer A and eluted with 1 mg/ml p65peptide. A similar experiment was performed with 10 mg³⁵S-metabolically-labeled HeLa cell lysate (100 μCi/ml Met/Cys for 8hours) and 25 μl p65-immunobeads. Eluate-fractions derived from both thehot and cold lysates were mixed, boiled in SDS-sample buffer andanalyzed by 7.5% SDS-PAGE and autoradiography. Gel slices correspondingto the autoradiogram signals were excised and their protein-bandssequenced by mass-spectrometry, as described below.

Three immunoaffinity-purified fractions were compared by SDS-pageanalysis (FIG. 7A): 1) a fraction containing IκBα/NF-κB complexes thatwas not phosphorylated by IKK2-EE, but incubated with HeLa lysate; 2) afraction subjected to IKK2-EE phosphorylation and subsequent incubationwith HeLa lysate; 3) a fraction phosphorylated by IKK2-EE, but notincubated with HeLa lysate. All incubations were performed onimmunobead-immobilized complexes, which were then extensively washed andeluted with the p65 peptide.

SDS-PAGE analysis of the three fractions revealed pattern-changes due toIKK phosphorylation or to further immuno-adsorption of IκBα/NF-κBproteins, but did not discern any protein recruited to the IκB-complexfollowing IKK phosphorylation. The complexity of the protein stainingcould obscure the presence of any recruited protein migrating along withan immunopurified protein. To identify the recruited protein,mass-spectrometry analysis was performed on a dozen ColloidalBlue-stained bands derived from fractions 1 and 2. This analysisrevealed the presence of nearly the full spectrum of the Rel familyproteins and IκBα: NF-κB1 (p105), NF-κB2 (p100), RelA (p65), p50, p49,C-Rel, IκBα and IκBε. Only a few other proteins wereco-immunoprecipitated with the IκB/NF-κB complex, particularlyGRP78/Bip, Hsp 70 and Hsc 70.

To circumvent the possible masking of the putative pIκB-ubiquitinligase, we replaced the ligase source with ³⁵S-biosynthetically-labeledHeLa lysate and traced the IκBα-associated proteins by SDS-PAGE analysisand autoradiography (FIG. 7B). In parallel, the various fractions weretested for their ubiquitin-ligase capacity. The band-pattern of theactive fraction (lane 2) was compared with that of the non-active one(lane 1). Four ³⁵S-protein bands with a molecular mass of 54, 58, 61 and64 kD were distinguished in lane 2. Some of these protein bands couldrepresent components of the ubiquitin ligase that recognizes pIκBαdirectly whereas others might have associated with pIκBα indirectly orwith another component of the IKK-phosphorylated complex. To sort outthe ligase component that recognizes pIκBα directly, pp12 or the controlpeptide p12S/E was added to the radiolabeled HeLa lysate, which was thenincubated with the immuno-bound IκBα/NF-κB complex. A comparison of theeluted fractions showed that of the four distinctive bands present onlyin fraction 2, three bands were eliminated by the specific pp12 peptide(p54, p58 and p61), whereas only the 64 kD band persisted in thepresence of pp12 (FIG. 7B, compare lanes 2 and 3). The control peptidedid not affect the association of any of the distinctive proteins withpIκBα (lane 4). Two of the pIκBα interacting proteins, p58 and p54, wereconsistently present and always associated with the specificubiquitin-ligase activity.

Example 5 Identification of Human E3 Ubiquitin Ligase

This Example illustrates the isolation and characterization of human E3ubiquitin ligase.

The 54 and 58 kD bands described in the previous Example were excisedfrom a ligase-positive and a ligase-negative (HeLa lysate incubated witha non-phosphorylated IκBα-complex) lane, the proteins digested in situ(Shevchenko et al., Anal. Chem. 68:850-858, 1996) and the trypticpeptides thus obtained were sequenced by nanoelectrospray massspectrometry (Wilm et al., Nature 379:466-469, 1996). Protein bands werereduced in-gel S-alkylated and digested in-gel with an excess of trypsin(overnight at room temperature) as described (Shevchenko et al., Anal.Chem. 68:850-858, 1996; Wilm et al., Nature 379:466-469, 1996). Piecesof gel were extracted and the resulting peptide mixtures wereconcentrated and desalted, using a micro-column containing 50 nl ofPoros R2 material (Perceptive Biosystems, Framingham, Mass.). Peptideswere eluted with 1 μl of 60% methanol, 5% formic acid directly into ananoelectrospray needle. Nanoelectrospray spectra were recorded on aquadrupole time-of-flight mass spectrometer (QqTOF, Perkin-Elmer Sciex,Toronto, Canada). Peptide sequence tags (Mann and Wilm, Anal. Chem.66:4390-4399, 1994) were assembled from fragmentation spectra andsearched against a non redundant protein sequence database (nrdb)maintained at the European BioInformatics Institute (EBI, Hinxton Park,England) using the program PeptideSearch (Mann and Wilm, Anal. Chem.66:4390-4399, 1994).

Mass spectra of the 54 kD gel band revealed a complex peptide mixture(FIG. 8A) from which several peptides were selected for fragmentation.Proteins identified by peptide sequence tag searching (Mann and Wilm,Anal. Chem. 66:4390-4399, 1994) included NF-κB1 (p50), IκB kinase α,IκBε, RelB, tubulin beta-1 chain, and thyroid receptor initiator bindingprotein. To identify the protein associated with the E3 activity,additional peptides, present in small amounts, were selected forsequencing by comparing the spectrum of the 54 kD bands from the activefraction with that of a similar band from the non-active one (FIG. 8B).The peptide sequence tag (1587.81) VVNV (SEQ ID NO:23) (1999.09) wasderived from the fragmentation spectrum shown in FIG. 8C andunambiguously identified as AAVNVVDFDDKYIVSAS (SEQ ID NO:24). Furtherspectra identified the peptides LEGHEELVR (SEQ ID NO:25), LVVSGSSDNTIR(SEQ ID NO:26), IQDIETIESNWR (SEQ ID NO:27) and VISEGMLWK (SEQ IDNO:28). The first four fragments have sequences present within the humanF-box/WD protein β-TrCP (Margottin et al., Mol. Cell 1:565-574, 1998).However, the fifth peptide (VISEGMLWK (SEQ ID NO:28)) matches that of apeptide from the Drosophila Slimb protein (see Jiang and Struhl, Nature391:493-496, 1998), which is highly homologous to human β-TrCP. Furthersequencing identified the human E3 ubiquitin ligase nucleotide sequenceprovided in FIG. 9 (SEQ ID NO:15), and the predicted protein sequenceprovided in FIG. 10 (SEQ ID NO:16). Thus, the human E3 ubiquitin ligaseappears to be a novel member of the β-TrCP/Slimb family of homologousproteins.

Example 6 Further Characterization of E3 Ubiquitin Ligase Activity

This Example further illustrates the ubiquitin ligase activity of thehuman E3 ubiquitin ligase family members β-TrCP and Slimb.

The ability of these proteins to bind pIκBα specifically and assist inits ubiquitination was examined in a cell-free system. The IκBα/NF-κBcomplex was immunopurified from HeLa cells and the immune complex waseither phosphorylated with IKK2-EE or mock-phosphorylated as describedabove. It was then incubated with the following immobilized FLAG-taggedE3 family members immunoprecipitated from transfected 293 cells: mouseβ-TrCP (mβ-TrCP), human β-TrCP (hβ-TrCP), human β-TrCP with a deletionof the F box region residues 122-168 (Δβ-TrCP) and the Drosophila Slimbprotein. The bound material was analyzed by Western blotting withanti-IκBα and anti-FLAG antibodies. All of these proteins exclusivelybound IKK-phosphorylated, but not mock-phosphorylated, IκBα (see FIG.11A). However, the human and mouse β-TrCP bound IκBα far better than thehighly homologous Drosophila protein (compare lanes 2, 4, 6 and 8).Δβ-TrCP bound pIκBα even better than the wild type protein, indicatingthat the F-box region was dispensable for binding. Furthermore, β-TrCPbinding was abrogated by a peptide representing the pIκBα recognitionmotif (pp10; DRHDS(PO₃)GLDS(PO₃)M (SEQ ID NO:29); see FIG. 11B, lane 3),but not by the control peptide (lane 4), specifying the site of pIκBαrecognition of the conserved DS(PO₃)GLDS(PO₃) (SEQ ID NO:30) sequence.

To evaluate the effect of binding on ubiquitination, the E3 familymembers and the deletion mutant were used as a source of E3 activity inpIκBα ubiquitination. In the presence of E1 and E2 (UBC5C), the wildtype β-TrCP proteins facilitated the ubiquitination of pIκBα, but not ofthe non-phosphorylated IκBα (see FIG. 11C, lanes 1-4). Δβ-TrCP, devoidof the F-box protein-protein interaction module, failed to promoteubiquitination (lanes 7 and 8), in spite of its binding capacity (FIG.11A, lane 6). Although Slimb facilitated some pIκBα ubiquitination, itwas at least ten-fold less efficient than the human and mouse β-TrCP(based on similar FLAG-tag expression levels), corresponding to itsweaker activity.

The modular design of these family members and the in vitro analysisdescribed herein suggested that deletion of the F-box would result in aprotein that functions as a dominant negative molecule in vivo. In fact,transient over-expression of the Δβ-TrCP inhibited the degradation ofendogenous IκBα in stimulated Jurkat cells, resulting in accumulation ofpIκBα (FIG. 12A). Consequently, activation of NF-κB was inhibited (FIG.12B). NF-κB activation was specific, as Δβ-TrCP did not affectactivation of an NF-AT reporter. Of note is the fact that NF-κBinhibition was also observed with wild type Slimb, whereas theexpression of wild type human β-TrCP was not inhibitory (FIG. 12B).Therefore, overexpression of wild type Slimb has a dominant negativeeffect on NF-κB activation, probably linked to its relatively poor pIκBαubiquitination activity (FIG. 11B).

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

SUMMARY OF SEQUENCE LISTING

SEQ ID NO:1 is amino acid sequence of IκBα

SEQ ID NO:2 is DNA sequence of IκBα

SEQ ID NO:3 is amino acid sequence of IκBβ

SEQ ID NO:4 is DNA sequence of IκBβ

SEQ ID NO:5 is amino acid sequence of pp7

SEQ ID NO:6 is amino acid sequence of pp11

SEQ ID NO:7 is amino acid sequence of pp15

SEQ ID NO:8 is amino acid sequence of pp19

SEQ ID NO:9 is amino acid sequence of pp21

SEQ ID NO:10 is amino acid sequence of phospho-Fos peptide

SEQ ID NO:11 is amino acid sequence of pp21 S/A

SEQ ID NO:12 is amino acid sequence of HA-tagged IκBα

SEQ ID NO:13 is amino acid sequence of HA-tagged S32, 36 IκBα

SEQ ID NO:14 is amino acid sequence of HA-tagged IκBβ

SEQ ID NO:15 is DNA sequence of human E3 ubiquitin ligase

SEQ ID NO:16 is predicted amino acid sequence of human E3 ubiquitinligase

SEQ ID NO:17 is DNA sequence of human β-TrCP

SEQ ID NO:18 is amino acid sequence of human E3 β-TrCP

SEQ ID NO:19 is phosphorylation site of IκBα

SEQ ID NO:20 is retrieved β-TrCP sequence

SEQ ID NO:21 is amino acid sequence of cognate p64 peptide

SEQ ID NO:22 is amino acid sequence of pIκBα peptide pp12

SEQ ID NO:23 is peptide sequence tag of human E3 ubiquitin ligase

SEQ ID NO:24 is peptide from human E3 ubiquitin ligase

SEQ ID NO:25 is peptide from human E3 ubiquitin ligase

SEQ ID NO:26 is peptide from human E3 ubiquitin ligase

SEQ ID NO:27 is peptide from human E3 ubiquitin ligase

SEQ ID NO:28 is peptide from human E3 ubiquitin ligase

SEQ ID NO:29 is amino acid sequence of pIκBα recognition motif

SEQ ID NO:30 is conserved pIκBα sequence

30 1 317 PRT Homo sapiens 1 Met Phe Gln Ala Ala Glu Arg Pro Gln Glu TrpAla Met Glu Gly Pro 1 5 10 15 Arg Asp Gly Leu Lys Lys Glu Arg Leu LeuAsp Asp Arg His Asp Ser 20 25 30 Gly Leu Asp Ser Met Lys Asp Glu Glu TyrGlu Gln Met Val Lys Glu 35 40 45 Leu Gln Glu Ile Arg Leu Glu Pro Gln GluVal Pro Arg Gly Ser Glu 50 55 60 Pro Trp Lys Gln Gln Leu Thr Glu Asp GlyAsp Ser Phe Leu His Leu 65 70 75 80 Ala Ile Ile His Glu Glu Lys Ala LeuThr Met Glu Val Ile Arg Gln 85 90 95 Val Lys Gly Asp Leu Ala Phe Leu AsnPhe Gln Asn Asn Leu Gln Gln 100 105 110 Thr Pro Leu His Leu Ala Val IleThr Asn Gln Pro Glu Ile Ala Glu 115 120 125 Ala Leu Leu Gly Ala Gly CysAsp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140 Asn Thr Pro Leu His LeuAla Cys Glu Gln Gly Cys Leu Ala Ser Val 145 150 155 160 Gly Val Leu ThrGln Ser Cys Thr Thr Pro His Leu His Ser Ile Leu 165 170 175 Lys Ala ThrAsn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile 180 185 190 His GlyTyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp 195 200 205 ValAsn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230235 240 Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly GlnLeu 260 265 270 Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp GluGlu Ser 275 280 285 Tyr Asp Thr Glu Ser Glu Phe Thr Glu Phe Thr Glu AspGlu Leu Pro 290 295 300 Tyr Asp Asp Cys Val Phe Gly Gly Gln Arg Leu ThrLeu 305 310 315 2 1550 DNA Homo sapiens 2 tgccgccgtc ccgcccgccagcgccccagc gaggaagcag cgcgcagccc gcggcccagc 60 gcacccgcag cagcgcccgcagctcgtccg cgccatgttc caggcggccg agcgccccca 120 ggagtgggcc atggagggcccccgcgacgg gctgaagaag gagcggctac tggacgaccg 180 ccacgacagc ggcctggactccatgaaaga cgaggagtac gagcagatgg tcaaggagct 240 gcaggagatc cgcctcgagccgcaggaggt gccgcgcggc tcggagccct ggaagcagca 300 gctcaccgag gacggggactcgttcctgca cttggccatc atccatgaag aaaaggcact 360 gaccatggaa gtgatccgccaggtgaaggg agacctggct ttcctcaact tccagaacaa 420 cctgcagcag actccactccacttggctgt gatcaccaac cagccagaaa ttgctgaggc 480 acttctggga gctggctgtgatcctgagct ccgagacttt cgaggaaata cccccctaca 540 ccttgcctgt gagcagggctgcctggccag cgtgggagtc ctgactcagt cctgcaccac 600 cccgcacctc cactccatcctgaaggctac caactacaat ggccacacgt gtctacactt 660 agcctctatc catggctacctgggcatcgt ggagcttttg gtgtccttgg gtgctgatgt 720 caatgctcag gagccctgtaatggccggac tgcccttcac ctcgcagtgg acctgcaaaa 780 tcctgacctg gtgtcactcctgttgaagtg tggggctgat gtcaacagag ttacctacca 840 gggctattct ccctaccagctcacctgggg ccgcccaagc acccggatac agcagcagct 900 gggccagctg acactagaaaaccttcagat gctgccagag agtgaggatg aggagagcta 960 tgacacagag tcagagttcacggagttcac agaggacgag ctgccctatg atgactgtgt 1020 gtttggaggc cagcgtctgacgttatgagt gcaaaggggc tgaaagaaca tggacttgta 1080 tatttgtaca aaaaaaaagttttatttttc taaaaaaaga aaaaagaaga aaaaatttaa 1140 agggtgtact tatatccacactgcacactg cctagcccaa aacgtcttat tgtggtagga 1200 tcagccctca ttttgttgcttttgtgaact ttttgtaggg gacgagaaag atcattgaaa 1260 ttctgagaaa acttcttttaaacctcacct ttgtggggtt tttggagaag gttatcaaaa 1320 atttcatgga aggaccacattttatattta ttgtgcttcg agtgactgac cccagtggta 1380 tcctgtgaca tgtaacagccaggagtgtta agcgttcagt gatgtggggt gaaaagttac 1440 tacctgtcaa ggtttgtgttaccctcctgt aaatggtgta cataatgtat tgttggtaat 1500 tattttggta cttttatgatgtatatttat taaagagatt tttacaaatg 1550 3 359 PRT Homo sapiens 3 Met AlaGly Val Ala Cys Leu Gly Lys Thr Ala Asp Ala Asp Glu Trp 1 5 10 15 CysAsp Ser Gly Leu Gly Ser Leu Gly Pro Asp Ala Ala Ala Pro Gly 20 25 30 GlyPro Gly Leu Gly Ala Glu Leu Gly Pro Glu Leu Ser Trp Ala Pro 35 40 45 LeuVal Phe Gly Tyr Val Thr Glu Asp Gly Asp Thr Ala Leu His Leu 50 55 60 AlaVal Ile His Gln His Glu Pro Phe Leu Asp Phe Leu Leu Gly Phe 65 70 75 80Ser Ala Gly His Glu Tyr Leu Asp Leu Gln Asn Asp Leu Gly Gln Thr 85 90 95Ala Leu His Leu Ala Ala Ile Leu Gly Glu Ala Ser Thr Val Glu Lys 100 105110 Leu Tyr Ala Ala Gly Ala Gly Val Leu Val Ala Glu Arg Gly Gly His 115120 125 Thr Ala Leu His Leu Ala Cys Arg Val Arg Ala His Thr Cys Ala Cys130 135 140 Val Leu Leu Gln Pro Arg Pro Ser His Pro Arg Asp Ala Ser AspThr 145 150 155 160 Tyr Leu Thr Gln Ser Gln Asp Cys Thr Pro Asp Thr SerHis Ala Pro 165 170 175 Ala Ala Val Asp Ser Gln Pro Asn Pro Glu Asn GluGlu Glu Pro Arg 180 185 190 Asp Glu Asp Trp Arg Leu Gln Leu Glu Ala GluAsn Tyr Asp Gly His 195 200 205 Thr Pro Leu His Val Ala Val Ile His LysAsp Ala Glu Met Val Arg 210 215 220 Leu Leu Arg Asp Ala Gly Ala Asp LeuAsn Lys Pro Glu Pro Thr Cys 225 230 235 240 Gly Arg Thr Pro Leu His LeuAla Val Glu Ala Gln Ala Ala Ser Val 245 250 255 Leu Glu Leu Leu Leu LysAla Gly Ala Asp Pro Thr Ala Arg Met Tyr 260 265 270 Gly Gly Arg Thr ProLeu Gly Ser Ala Leu Leu Arg Pro Asn Pro Ile 275 280 285 Leu Ala Arg LeuLeu Arg Ala His Gly Ala Pro Glu Pro Glu Asp Glu 290 295 300 Asp Asp LysLeu Ser Pro Cys Ser Ser Ser Gly Ser Asp Ser Asp Ser 305 310 315 320 AspAsn Arg Asp Glu Gly Asp Glu Tyr Asp Asp Ile Val Val His Ser 325 330 335Gly Arg Ser Gln Asn Arg Gln Pro Pro Ser Pro Ala Ser Lys Pro Leu 340 345350 Pro Asp Asp Pro Asn Pro Ala 355 4 1212 DNA Homo sapiens 4 gcgcactggagctcatcgca gagcccagcg acaggcaggc gaccacaggg ggccacccga 60 ggtggctggggccatggccg gggtcgcgtg cttggggaaa actgcggatg ccgatgaatg 120 gtgcgacagcggcctgggct ctctaggtcc cgacgcagcg gctcccggag gaccaggtct 180 gggcgcagagcttggcccag agctgtcgtg ggcgccctta gtctttggct acgtcactga 240 ggatggggacacagccctgc acttggctgt gattcatcag catgagccct tcctggattt 300 cctcctgggcttttccgccg gccacgagta ccttgacctg cagaatgacc taggccaaac 360 agccctgcatctagcagcca tccttgggga ggcatctaca gtagagaagt tgtatgcagc 420 cggtgcaggagtgttggtgg ctgagagagg gggccacacg gcattgcact tggcctgccg 480 ggtcagggcacacacgtgcg cgtgcgtact gctccagccc cgtcccagcc acccaagaga 540 tgcctcagatacctacctca ctcagagcca ggactgtacc ccagacacca gccatgcccc 600 tgctgccgtggattcccaac ccaacccaga gaacgaagag gagccgcgtg atgaagactg 660 gaggctacaactagaagctg aaaactatga tggccatacc ccactccatg tagctgtcat 720 ccacaaagatgcagagatgg tccggctgct cagggatgcc ggagccgacc tcaataaacc 780 ggagcctacgtgtggccgga cccctctgca cctggcagta gaagcccagg cagccagcgt 840 gctggaacttctcctgaaag ccggtgctga ccccaccgcc cgcatgtatg ggggccgcac 900 cccgcttggcagtgccctgc tccggcccaa ccccatcctt gcccgcctcc tccgtgcaca 960 tggggcccctgaacctgagg acgaggacga taagcttagc ccttgcagca gcagcggcag 1020 cgacagtgacagtgacaaca gagatgaggg cgatgaatat gatgacatcg tggttcacag 1080 tggcaggagccaaaaccgac aaccgccttc cccggcatcc aaacctcttc ctgatgaccc 1140 caaccctgcctgacttaagt gctaatatta atataatttc caacttaata aaattgcaga 1200 cctgacaaccag 1212 5 8 PRT Homo sapiens 5 Cys Asp Ser Gly Leu Asp Ser Met 1 5 6 12PRT Homo sapiens 6 Cys Asp Asp Arg His Asp Ser Gly Leu Asp Ser Met 1 510 7 16 PRT Homo sapiens 7 Cys Asp Asp Arg His Asp Ser Gly Leu Asp SerMet Lys Asp Glu Glu 1 5 10 15 8 20 PRT Homo sapiens 8 Cys Glu Arg LeuLeu Asp Asp Arg His Asp Ser Gly Leu Asp Ser Met 1 5 10 15 Lys Asp GluGlu 20 9 22 PRT Homo sapiens 9 Cys Lys Lys Glu Arg Leu Leu Asp Asp ArgHis Asp Ser Gly Leu Asp 1 5 10 15 Ser Met Lys Asp Glu Glu 20 10 19 PRTHomo sapiens 10 Ile Gly Arg Arg Gly Lys Val Glu Gln Leu Ser Pro Glu GluGlu Glu 1 5 10 15 Lys Arg Arg 11 22 PRT Homo sapiens 11 Cys Lys Lys GluArg Leu Leu Asp Asp Arg His Asp Ala Gly Leu Asp 1 5 10 15 Ala Met LysAsp Glu Glu 20 12 347 PRT Homo sapiens 12 Met Tyr Pro Tyr Asp Val ProAsp Tyr Ala Met Tyr Pro Tyr Asp Val 1 5 10 15 Pro Asp Tyr Ala Met TyrPro Tyr Asp Val Pro Asp Tyr Ala Met Phe 20 25 30 Gln Ala Ala Glu Arg ProGln Glu Trp Ala Met Glu Gly Pro Arg Asp 35 40 45 Gly Leu Lys Lys Glu ArgLeu Leu Asp Asp Arg His Asp Ser Gly Leu 50 55 60 Asp Ser Met Lys Asp GluGlu Tyr Glu Gln Met Val Lys Glu Leu Gln 65 70 75 80 Glu Ile Arg Leu GluPro Gln Glu Val Pro Arg Gly Ser Glu Pro Trp 85 90 95 Lys Gln Gln Leu ThrGlu Asp Gly Asp Ser Phe Leu His Leu Ala Ile 100 105 110 Ile His Glu GluLys Ala Leu Thr Met Glu Val Ile Arg Gln Val Lys 115 120 125 Gly Asp LeuAla Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln Thr Pro 130 135 140 Leu HisLeu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu Ala Leu 145 150 155 160Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly Asn Thr 165 170175 Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val Gly Val 180185 190 Leu Thr Gln Ser Cys Thr Thr Pro His Leu His Ser Ile Leu Lys Ala195 200 205 Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile HisGly 210 215 220 Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala AspVal Asn 225 230 235 240 Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu HisLeu Ala Val Asp 245 250 255 Leu Gln Asn Pro Asp Leu Val Ser Leu Leu LeuLys Cys Gly Ala Asp 260 265 270 Val Asn Arg Val Thr Tyr Gln Gly Tyr SerPro Tyr Gln Leu Thr Trp 275 280 285 Gly Arg Pro Ser Thr Arg Ile Gln GlnGln Leu Gly Gln Leu Thr Leu 290 295 300 Glu Asn Leu Gln Met Leu Pro GluSer Glu Asp Glu Glu Ser Tyr Asp 305 310 315 320 Thr Glu Ser Glu Phe ThrGlu Phe Thr Glu Asp Glu Leu Pro Tyr Asp 325 330 335 Asp Cys Val Phe GlyGly Gln Arg Leu Thr Leu 340 345 13 347 PRT Homo sapiens 13 Met Tyr ProTyr Asp Val Pro Asp Tyr Ala Met Tyr Pro Tyr Asp Val 1 5 10 15 Pro AspTyr Ala Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Met Phe 20 25 30 Gln AlaAla Glu Arg Pro Gln Glu Trp Ala Met Glu Gly Pro Arg Asp 35 40 45 Gly LeuLys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ala Gly Leu 50 55 60 Asp AlaMet Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu Leu Gln 65 70 75 80 GluIle Arg Leu Glu Pro Gln Glu Val Pro Arg Gly Ser Glu Pro Trp 85 90 95 LysGln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu Ala Ile 100 105 110Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Ile Arg Gln Val Lys 115 120125 Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln Thr Pro 130135 140 Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu Ala Leu145 150 155 160 Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg GlyAsn Thr 165 170 175 Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala SerVal Gly Val 180 185 190 Leu Thr Gln Ser Cys Thr Thr Pro His Leu His SerIle Leu Lys Ala 195 200 205 Thr Asn Tyr Asn Gly His Thr Cys Leu His LeuAla Ser Ile His Gly 210 215 220 Tyr Leu Gly Ile Val Glu Leu Leu Val SerLeu Gly Ala Asp Val Asn 225 230 235 240 Ala Gln Glu Pro Cys Asn Gly ArgThr Ala Leu His Leu Ala Val Asp 245 250 255 Leu Gln Asn Pro Asp Leu ValSer Leu Leu Leu Lys Cys Gly Ala Asp 260 265 270 Val Asn Arg Val Thr TyrGln Gly Tyr Ser Pro Tyr Gln Leu Thr Trp 275 280 285 Gly Arg Pro Ser ThrArg Ile Gln Gln Gln Leu Gly Gln Leu Thr Leu 290 295 300 Glu Asn Leu GlnMet Leu Pro Glu Ser Glu Asp Glu Glu Ser Tyr Asp 305 310 315 320 Thr GluSer Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp 325 330 335 AspCys Val Phe Gly Gly Gln Arg Leu Thr Leu 340 345 14 389 PRT Homo sapiens14 Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Met Tyr Pro Tyr Asp Val 1 510 15 Pro Asp Tyr Ala Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Met Ala 2025 30 Gly Val Ala Cys Leu Gly Lys Thr Ala Asp Ala Asp Glu Trp Cys Asp 3540 45 Ser Gly Leu Gly Ser Leu Gly Pro Asp Ala Ala Ala Pro Gly Gly Pro 5055 60 Gly Leu Gly Ala Glu Leu Gly Pro Glu Leu Ser Trp Ala Pro Leu Val 6570 75 80 Phe Gly Tyr Val Thr Glu Asp Gly Asp Thr Ala Leu His Leu Ala Val85 90 95 Ile His Gln His Glu Pro Phe Leu Asp Phe Leu Leu Gly Phe Ser Ala100 105 110 Gly His Glu Tyr Leu Asp Leu Gln Asn Asp Leu Gly Gln Thr AlaLeu 115 120 125 His Leu Ala Ala Ile Leu Gly Glu Ala Ser Thr Val Glu LysLeu Tyr 130 135 140 Ala Ala Gly Ala Gly Val Leu Val Ala Glu Arg Gly GlyHis Thr Ala 145 150 155 160 Leu His Leu Ala Cys Arg Val Arg Ala His ThrCys Ala Cys Val Leu 165 170 175 Leu Gln Pro Arg Pro Ser His Pro Arg AspAla Ser Asp Thr Tyr Leu 180 185 190 Thr Gln Ser Gln Asp Cys Thr Pro AspThr Ser His Ala Pro Ala Ala 195 200 205 Val Asp Ser Gln Pro Asn Pro GluAsn Glu Glu Glu Pro Arg Asp Glu 210 215 220 Asp Trp Arg Leu Gln Leu GluAla Glu Asn Tyr Asp Gly His Thr Pro 225 230 235 240 Leu His Val Ala ValIle His Lys Asp Ala Glu Met Val Arg Leu Leu 245 250 255 Arg Asp Ala GlyAla Asp Leu Asn Lys Pro Glu Pro Thr Cys Gly Arg 260 265 270 Thr Pro LeuHis Leu Ala Val Glu Ala Gln Ala Ala Ser Val Leu Glu 275 280 285 Leu LeuLeu Lys Ala Gly Ala Asp Pro Thr Ala Arg Met Tyr Gly Gly 290 295 300 ArgThr Pro Leu Gly Ser Ala Leu Leu Arg Pro Asn Pro Ile Leu Ala 305 310 315320 Arg Leu Leu Arg Ala His Gly Ala Pro Glu Pro Glu Asp Glu Asp Asp 325330 335 Lys Leu Ser Pro Cys Ser Ser Ser Gly Ser Asp Ser Asp Ser Asp Asn340 345 350 Arg Asp Glu Gly Asp Glu Tyr Asp Asp Ile Val Val His Ser GlyArg 355 360 365 Ser Gln Asn Arg Gln Pro Pro Ser Pro Ala Ser Lys Pro LeuPro Asp 370 375 380 Asp Pro Asn Pro Ala 385 15 4230 DNA Homo sapiens 15gcgaggcggg gccgccgggg ccgccatgga gcccgactcg gtgattgagg acaagaccat 60cgagctcatg tgttctgtgc caaggtcttt gtggctaggc tgcgccaacc tggtagagag 120catgtgcgca ctgagttgcc tgcagagcat gcccagtgtc agatgtctcc agataagtaa 180tggaacatca tctgtgatcg tctccagaaa gaggccatca gaaggaaact atcaaaaaga 240aaaagacttg tgtattaaat attttgacca gtggtctgaa tcagatcaag tggaatttgt 300ggaacatctt atttcacgaa tgtgtcatta tcagcatgga catattaact cttacctgaa 360gcccatgttg cagcgggact ttattaccgc tttaccagag caaggcttag atcacatagc 420agaaaacatt ctttcgtacc tggatgccag gtctctgtgt gcagcagagc tggtatgtaa 480agaatggcag cgagtgatct cagaaggaat gctttggaag aagctgattg aacgaatggt 540acgcactgat cccctatgga aaggactttc agaaagaaga gggtgggatc agtacctgtt 600taaaaacaga cccacagatg gccctccaaa ttcattttat aggtcattat acccaaagat 660tatccaggat atagagacta tagaatctaa ctggcggtgt ggacgacaca acttgcagag 720gattcagtgc cgctctgaaa atagtaaagg tgtctactgt ttacagtacg atgatgaaaa 780aattatcagt ggcctacgag ataattctat taagatatgg gataaaacca gcctggaatg 840tttgaaagtg ttaacaggac acacaggctc tgtcctctgt ctgcagtatg atgagcgtgt 900cattgtaact ggctcttcag attctacggt gagagtgtgg gatgtgaaca cgggtgaagt 960tcttaacaca ttgatccacc acaatgaggc tgtattgcac ttacgcttca gcaatggact 1020gatggtgacc tgttccaagg accgctccat tgctgtgtgg gacatggctt ctgcgaccga 1080catcacttta cgccgtgtcc tggttggcca ccgggctgcc gtcaatgtag tagactttga 1140cgacaagtac atcgtgtctg cctctggtga caggaccatc aaagtctgga gcacgagcac 1200ctgtgaattt gttcgtactc tcaatgggca caagcggggc attgcctgtc tccagtacag 1260ggatcgcctg gttgttagtg gatcatcaga taataccatt aggctctggg atattgaatg 1320tggtgcctgt ttaagagtcc tagagggaca tgaagaattg gtccgatgca tccggtttga 1380taacaagagg attgtcagtg gggcctatga tgggaaaatt aaagtttggg acttgcaagc 1440tgctcttgac cctcgagccc cagcaagcac attgtgtttg cgcacattgg tggaacattc 1500tggacgtgtg tttcggctcc agtttgatga gtttcagatc atcagcagct cccatgatga 1560cactattttg atttgggatt tcttaaatgt gcctcccagt gcccagaatg agacccgttc 1620tccctccaga acatacactt acatctctag ataacagtct gcactttcac ccgtttcagg 1680gttttctagt cttgaactac tggctacgtg gctaccaaat gcctaaggga gttcgttcac 1740agctgagtta tgaagctgga attggttcta gacgctgggt agatgcaaag cagcctaact 1800cttcaagtac cgacatttct cacctctgat tccggctctc ctttgagaag gagaccttag 1860cttccccggc ttcaagtaga acagaagccc gtttccttcc ctcatcagtg aaaaaatcta 1920atgtttcaaa tgtaaattgt tcatagaaaa ggaacataga atctgtttta cagaagtaaa 1980tcgaccgtca agagaagact tggcctctaa tttatattgc tttgcacttt ggtttgatat 2040taagaaacag cattcttctt cagtgaaatt ttgggtgcca aacacctacc cagaatgtcc 2100agggctttca ttttcaaaag ttagcattct ccttttgacc gtccaagtca ttatgaattc 2160tgacttgttg tattaggaac atgttggaca gtggaaaatt ttctctggat tgttttagta 2220atatttttgg gattatactt cctttctgta ccaatttctt ttaatttaaa gaactataag 2280tcagttatat tatctaccaa caggtaatat agctctttct tttattaact gttctctgtc 2340ccccaaccat ctcctgatat ttggtagagt aacaccttta tacgtgtgct tgcctcctaa 2400tttaaaatac tgtattcgca tgtagatata atgtacataa cagtttaacc tcaaagttgc 2460tggagtcagg gccccctgtg cttgagacac taatacagag tgtgttcgca cttagccatg 2520ggctgggctc aagaacctga tacctgggtt gatgtggatt acctagaacc cttcctgcag 2580tattcataca gtgtttttat tttgttgttg tcattgcgtg tgtgtggttt gtgtgtgttt 2640ttaatgagaa tcttgtttta aaatgtaatt tctaaggttt aacaccaaaa tgttttattt 2700gttgtggagt atatattata caatagagag gtaccttaaa cattttttgt tcttattctt 2760tttctcataa gtactcctga gtacaagtgg tcacctccca tagtattcat ttggcttcgc 2820tgtcaaaaat cattattctg tgcagtcgtg gccctgggaa ggggaaataa gaaggccctg 2880ttgacgggct gtcttggctc tggaattcat gcatcctggc cttgccaagg ttctggcagg 2940gcctgctggt gtgttggagc ctgcagggca ggtcaggctg gttcagaggc ccatgctgag 3000tggtgtcagc tgctgctgtg gccccagcag gttctcaaag ctcccaagtc ctccctacga 3120cacagcccaa atgtgtaaat ggcactgttg ccctgacagt gcatggaaag gacgttggca 3180tccaattggc actccttctc ccttattcaa tattaggttt gatttgccct tcgccattgt 3240ttccaaagat caaggaatgt caataacatt ttaaaggacc aataaacagc ctcctataaa 3300gtaaacctct tcccgtggaa gcacactcta ctactaaagg gaaggcccct gggctctgat 3360ttgtcctttg cattgagaac ggtgtgggga tcagtgtgtg tgtatgtgat ttgtttattg 3420agttggcttt gcttttttag tttttctttt aaaaataaaa tccttccttc ccatgttact 3480aaattaattt atgtttttga gaggttgagt ctcaaagtgt aaacaataaa cctccattca 3540taaggtggat gttgtaagct tgatggtggt tgtgaaagtg atttagcttt gaccactttt 3600catcctacag cttcaatatc aaactggtta ggaaagccca gggggaaggg agggggcagg 3660ggaggaggca attctgaatg aatgaatgga ttttttgttg tttttgcatg tttaatatag 3720aagttcccct cgttccttgg gagatgatgg cctttgaata tgcagacaac ctttgaattg 3780tgcctactaa attatagcag gggactttgg cacccaagga gttctgactt tctgggatta 3840taatagtaat tcccagccat actctggact ttattttgct aaccataact gagcaaatgt 3900aaattactgc tatattaatg ttttaaagca ctgggatagt ctaattctaa cttgtaatta 3960attatgtttg ccaattatct gtttgaaata aatttgtgtc tgaacagcta ttgaaactgt 4020taaattgtac agatattatt catgacagct ttgtactgtg gaatgtgctt aataaaaaac 4080aaaaaagttt gacttttgtc cagtaaattg ctaagtaatg tcaataaatc gagtatgggt 4140attatgcagt gcacctaatc tggcttcatg caattgttac ttcagctact gattcaaagc 4200caatactctt aataaagtgt tgcaatactc 4230 16 542 PRT Homo sapiens 16 Met GluPro Asp Ser Val Ile Glu Asp Lys Thr Ile Glu Leu Met Cys 1 5 10 15 SerVal Pro Arg Ser Leu Trp Leu Gly Cys Ala Asn Leu Val Glu Ser 20 25 30 MetCys Ala Leu Ser Cys Leu Gln Ser Met Pro Ser Val Arg Cys Leu 35 40 45 GlnIle Ser Asn Gly Thr Ser Ser Val Ile Val Ser Arg Lys Arg Pro 50 55 60 SerGlu Gly Asn Tyr Gln Lys Glu Lys Asp Leu Cys Ile Lys Tyr Phe 65 70 75 80Asp Gln Trp Ser Glu Ser Asp Gln Val Glu Phe Val Glu His Leu Ile 85 90 95Ser Arg Met Cys His Tyr Gln His Gly His Ile Asn Ser Tyr Leu Lys 100 105110 Pro Met Leu Gln Arg Asp Phe Ile Thr Ala Leu Pro Glu Gln Gly Leu 115120 125 Asp His Ile Ala Glu Asn Ile Leu Ser Tyr Leu Asp Ala Arg Ser Leu130 135 140 Cys Ala Ala Glu Leu Val Cys Lys Glu Trp Gln Arg Val Ile SerGlu 145 150 155 160 Gly Met Leu Trp Lys Lys Leu Ile Glu Arg Met Val ArgThr Asp Pro 165 170 175 Leu Trp Lys Gly Leu Ser Glu Arg Arg Gly Trp AspGln Tyr Leu Phe 180 185 190 Lys Asn Arg Pro Thr Asp Gly Pro Pro Asn SerPhe Tyr Arg Ser Leu 195 200 205 Tyr Pro Lys Ile Ile Gln Asp Ile Glu ThrIle Glu Ser Asn Trp Arg 210 215 220 Cys Gly Arg His Asn Leu Gln Arg IleGln Cys Arg Ser Glu Asn Ser 225 230 235 240 Lys Gly Val Tyr Cys Leu GlnTyr Asp Asp Glu Lys Ile Ile Ser Gly 245 250 255 Leu Arg Asp Asn Ser IleLys Ile Trp Asp Lys Thr Ser Leu Glu Cys 260 265 270 Leu Lys Val Leu ThrGly His Thr Gly Ser Val Leu Cys Leu Gln Tyr 275 280 285 Asp Glu Arg ValIle Val Thr Gly Ser Ser Asp Ser Thr Val Arg Val 290 295 300 Trp Asp ValAsn Thr Gly Glu Val Leu Asn Thr Leu Ile His His Asn 305 310 315 320 GluAla Val Leu His Leu Arg Phe Ser Asn Gly Leu Met Val Thr Cys 325 330 335Ser Lys Asp Arg Ser Ile Ala Val Trp Asp Met Ala Ser Ala Thr Asp 340 345350 Ile Thr Leu Arg Arg Val Leu Val Gly His Arg Ala Ala Val Asn Val 355360 365 Val Asp Phe Asp Asp Lys Tyr Ile Val Ser Ala Ser Gly Asp Arg Thr370 375 380 Ile Lys Val Trp Ser Thr Ser Thr Cys Glu Phe Val Arg Thr LeuAsn 385 390 395 400 Gly His Lys Arg Gly Ile Ala Cys Leu Gln Tyr Arg AspArg Leu Val 405 410 415 Val Ser Gly Ser Ser Asp Asn Thr Ile Arg Leu TrpAsp Ile Glu Cys 420 425 430 Gly Ala Cys Leu Arg Val Leu Glu Gly His GluGlu Leu Val Arg Cys 435 440 445 Ile Arg Phe Asp Asn Lys Arg Ile Val SerGly Ala Tyr Asp Gly Lys 450 455 460 Ile Lys Val Trp Asp Leu Gln Ala AlaLeu Asp Pro Arg Ala Pro Ala 465 470 475 480 Ser Thr Leu Cys Leu Arg ThrLeu Val Glu His Ser Gly Arg Val Phe 485 490 495 Arg Leu Gln Phe Asp GluPhe Gln Ile Ile Ser Ser Ser His Asp Asp 500 505 510 Thr Ile Leu Ile TrpAsp Phe Leu Asn Val Pro Pro Ser Ala Gln Asn 515 520 525 Glu Thr Arg SerPro Ser Arg Thr Tyr Thr Tyr Ile Ser Arg 530 535 540 17 2151 DNA Homosapiens 17 tgcgttggct gcggcctggc accaaagggg cggccccggc ggagagcggacccagtggcc 60 tcggcgatta tggacccggc cgaggcggtg ctgcaagaga aggcactcaagtttatgaat 120 tcctcagaga gagaagactg taataatggc gaacccccta ggaagataataccagagaag 180 aattcactta gacagacata caacagctgt gccagactct gcttaaaccaagaaacagta 240 tgtttagcaa gcactgctat gaagactgag aattgtgtgg ccaaaacaaaacttgccaat 300 ggcacttcca gtatgattgt gcccaagcaa cggaaactct cagcaagctatgaaaaggaa 360 aaggaactgt gtgtcaaata ctttgagcag tggtcagagt cagatcaagtggaatttgtg 420 gaacatctta tatcccaaat gtgtcattac caacatgggc acataaactcgtatcttaaa 480 cctatgttgc agagagattt cataactgct ctgccagctc ggggattggatcatatcgct 540 gagaacattc tgtcatacct ggatgccaaa tcactatgtg ctgctgaacttgtgtgcaag 600 gaatggtacc gagtgacctc tgatggcatg ctgtggaaga agcttatcgagagaatggtc 660 aggacagatt ctctgtggag aggcctggca gaacgaagag gatggggacagtatttattc 720 aaaaacaaac ctcctgacgg gaatgctcct cccaactctt tttatagagcactttatcct 780 aaaattatac aagacattga gacaatagaa tctaattgga gatgtggaagacatagttta 840 cagagaattc actgccgaag tgaaacaagc aaaggagttt actgtttacagtatgatgat 900 cagaaaatag taagcggcct tcgagacaac acaatcaaga tctgggataaaaacacattg 960 gaatgcaagc gaattctcac aggccataca ggttcagtcc tctgtctccagtatgatgag 1020 agagtgatca taacaggatc atcggattcc acggtcagag tgtgggatgtaaatacaggt 1080 gaaatgctaa acacgttgat tcaccattgt gaagcagttc tgcacttgcgtttcaataat 1140 ggcatgatgg tgacctgctc caaagatcgt tccattgctg tatgggatatggcctcccca 1200 actgacatta ccctccggag ggtgctggtc ggacaccgag ctgctgtcaatgttgtagac 1260 tttgatgaca agtacattgt ttctgcatct ggggatagaa ctataaaggtatggaacaca 1320 agtacttgtg aatttgtaag gaccttaaat ggacacaaac gaggcattgcctgtttgcag 1380 tacagggaca ggctggtagt gagtggctca tctgacaaca ctatcagattatgggacata 1440 gaatgtggtg catgtttacg agtgttagaa ggccatgagg aattggtgcgttgtattcga 1500 tttgataaca agaggatagt cagtggggcc tatgatggaa aaattaaagtgtgggatctt 1560 gtggctgctt tggacccccg tgctcctgca gggacactct gtctacggacccttgtggag 1620 cattccggaa gagtttttcg actacagttt gatgaattcc agattgtcagtagttcacat 1680 gatgacacaa tcctcatctg ggacttccta aatgatccag ctgcccaagctgaacccccc 1740 cgttcccctt ctcgaacata cacctacatc tccagataaa taaccatacactgacctcat 1800 acttgcccag gacccattaa agttgcggta tttaacgtat ctgccaataccaggatgagc 1860 aacaacagta acaatcaaac tactgcccag tttccctgga ctagccgaggagcagggctt 1920 tgagactcct gttgggacac agttggtctg cagtcggccc aggacggtctactcagcaca 1980 actgactgct tcagtgctgc tatcagaaga tgtcttctat caattgtgaatgattggaac 2040 ttttaaacct cccctcctct cctcctttca cctctgcacc tagttttttcccattggttc 2100 cagacaaagg tgacttataa atatatttag tgttttgcca gaaaaaaaaa a2151 18 569 PRT Homo sapiens 18 Met Asp Pro Ala Glu Ala Val Leu Gln GluLys Ala Leu Lys Phe Met 1 5 10 15 Asn Ser Ser Glu Arg Glu Asp Cys AsnAsn Gly Glu Pro Pro Arg Lys 20 25 30 Ile Ile Pro Glu Lys Asn Ser Leu ArgGln Thr Tyr Asn Ser Cys Ala 35 40 45 Arg Leu Cys Leu Asn Gln Glu Thr ValCys Leu Ala Ser Thr Ala Met 50 55 60 Lys Thr Glu Asn Cys Val Ala Lys ThrLys Leu Ala Asn Gly Thr Ser 65 70 75 80 Ser Met Ile Val Pro Lys Gln ArgLys Leu Ser Ala Ser Tyr Glu Lys 85 90 95 Glu Lys Glu Leu Cys Val Lys TyrPhe Glu Gln Trp Ser Glu Ser Asp 100 105 110 Gln Val Glu Phe Val Glu HisLeu Ile Ser Gln Met Cys His Tyr Gln 115 120 125 His Gly His Ile Asn SerTyr Leu Lys Pro Met Leu Gln Arg Asp Phe 130 135 140 Ile Thr Ala Leu ProAla Arg Gly Leu Asp His Ile Ala Glu Asn Ile 145 150 155 160 Leu Ser TyrLeu Asp Ala Lys Ser Leu Cys Ala Ala Glu Leu Val Cys 165 170 175 Lys GluTrp Tyr Arg Val Thr Ser Asp Gly Met Leu Trp Lys Lys Leu 180 185 190 IleGlu Arg Met Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala Glu 195 200 205Arg Arg Gly Trp Gly Gln Tyr Leu Phe Lys Asn Lys Pro Pro Asp Gly 210 215220 Asn Ala Pro Pro Asn Ser Phe Tyr Arg Ala Leu Tyr Pro Lys Ile Ile 225230 235 240 Gln Asp Ile Glu Thr Ile Glu Ser Asn Trp Arg Cys Gly Arg HisSer 245 250 255 Leu Gln Arg Ile His Cys Arg Ser Glu Thr Ser Lys Gly ValTyr Cys 260 265 270 Leu Gln Tyr Asp Asp Gln Lys Ile Val Ser Gly Leu ArgAsp Asn Thr 275 280 285 Ile Lys Ile Trp Asp Lys Asn Thr Leu Glu Cys LysArg Ile Leu Thr 290 295 300 Gly His Thr Gly Ser Val Leu Cys Leu Gln TyrAsp Glu Arg Val Ile 305 310 315 320 Ile Thr Gly Ser Ser Asp Ser Thr ValArg Val Trp Asp Val Asn Thr 325 330 335 Gly Glu Met Leu Asn Thr Leu IleHis His Cys Glu Ala Val Leu His 340 345 350 Leu Arg Phe Asn Asn Gly MetMet Val Thr Cys Ser Lys Asp Arg Ser 355 360 365 Ile Ala Val Trp Asp MetAla Ser Pro Thr Asp Ile Thr Leu Arg Arg 370 375 380 Val Leu Val Gly HisArg Ala Ala Val Asn Val Val Asp Phe Asp Asp 385 390 395 400 Lys Tyr IleVal Ser Ala Ser Gly Asp Arg Thr Ile Lys Val Trp Asn 405 410 415 Thr SerThr Cys Glu Phe Val Arg Thr Leu Asn Gly His Lys Arg Gly 420 425 430 IleAla Cys Leu Gln Tyr Arg Asp Arg Leu Val Val Ser Gly Ser Ser 435 440 445Asp Asn Thr Ile Arg Leu Trp Asp Ile Glu Cys Gly Ala Cys Leu Arg 450 455460 Val Leu Glu Gly His Glu Glu Leu Val Arg Cys Ile Arg Phe Asp Asn 465470 475 480 Lys Arg Ile Val Ser Gly Ala Tyr Asp Gly Lys Ile Lys Val TrpAsp 485 490 495 Leu Val Ala Ala Leu Asp Pro Arg Ala Pro Ala Gly Thr LeuCys Leu 500 505 510 Arg Thr Leu Val Glu His Ser Gly Arg Val Phe Arg LeuGln Phe Asp 515 520 525 Glu Phe Gln Ile Val Ser Ser Ser His Asp Asp ThrIle Leu Ile Trp 530 535 540 Asp Phe Leu Asn Asp Pro Ala Ala Gln Ala GluPro Pro Arg Ser Pro 545 550 555 560 Ser Arg Thr Tyr Thr Tyr Ile Ser Arg565 19 6 PRT Homo sapiens 19 Asp Ser Gly Leu Asp Ser 1 5 20 20 PRT Homosapiens 20 Ala Ala Val Asn Val Val Asp Phe Asp Asp Lys Tyr Ile Val SerAla 1 5 10 15 Ser Gly Asp Arg 20 21 17 PRT Homo sapiens 21 Glu Leu PhePro Leu Ile Phe Pro Ala Glu Pro Ala Gln Ala Ser Gly 1 5 10 15 Pro 22 10PRT Homo sapiens MOD_RES (6) PHOSPHORYLATION 22 Cys Asp Arg His Asp SerGly Leu Asp Ser 1 5 10 23 4 PRT Homo sapiens 23 Val Val Asn Val 1 24 17PRT Homo sapiens 24 Ala Ala Val Asn Val Val Asp Phe Asp Asp Lys Tyr IleVal Ser Ala 1 5 10 15 Ser 25 9 PRT Homo sapiens 25 Leu Glu Gly His GluGlu Leu Val Arg 1 5 26 12 PRT Homo sapiens 26 Leu Val Val Ser Gly SerSer Asp Asn Thr Ile Arg 1 5 10 27 12 PRT Homo sapiens 27 Ile Gln Asp IleGlu Thr Ile Glu Ser Asn Trp Arg 1 5 10 28 9 PRT Homo sapiens 28 Val IleSer Glu Gly Met Leu Trp Lys 1 5 29 10 PRT Homo sapiens MOD_RES (5)PHOSPHORYLATION 29 Asp Arg His Asp Ser Gly Leu Asp Ser Met 1 5 10 30 6PRT Homo sapiens MOD_RES (2) PHOSPHORYLATION 30 Asp Ser Gly Leu Asp Ser1 5

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:16or a truncated portion thereof of at least 50 amino acid residueswherein said portion retains the ability to enhance ubiquitination ofphosphorylated IκB.
 2. An isolated polypeptide comprising SEQ ID NO:16or a truncated portion thereof of at least 200 amino acid residueswherein said portion retains the ability to enhance ubiquitination ofphosphorylated IκB.
 3. An isolated polypeptide comprising a variant ofSEQ ID NO:16 that differs therefrom at no more than 15% of the aminoacid residues of SEQ ID NO:16 wherein said variant retains the abilityto enhance ubiquitination of phosphorylated IκB.
 4. An isolatedpolypeptide comprising a variant of SEQ ID NO:16 that differs therefromat no more than 10% of the amino acid residues of SEQ ID NO:16 whereinsaid variant retains the ability to enhance ubiquitination ofphosphorylated IκB.
 5. An isolated polypeptide comprising a variant ofSEQ ID NO:16 that differs therefrom by deletion of amino acid residues122-168 of SEQ ID NO:16 or a truncated portion of said variant whereinsaid portion retains the ability to bind to phosphorylated IκB andinhibits ubiquitination of phosphorylated IκB.
 6. An isolatedpolypeptide comprising a truncated portion of SEQ ID NO:16 comprisingfrom 50 to 250 residues of SEQ ID NO:16 wherein said portion retains theability to bind to phosphorylated IκB and inhibits ubiquitination ofphosphorylated IκB.
 7. An isolated polypeptide comprising a truncatedportion of SEQ ID NO:16 comprising from 10 to 374 residues of SEQ IDNO:16 wherein said portion retains the ability to bind to phosphorylatedIκB and inhibits ubiquitination of phosphorylated IκB.